<?xml version="1.0" encoding="utf-8"?>
<InstanceReport xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xsd="http://www.w3.org/2001/XMLSchema">
  <Version>1.0.0.3</Version>
  <hasSegments>false</hasSegments>
  <ReportName>Employee Separation Costs (Unaudited)</ReportName>
  <RoundingOption />
  <Columns>
    <Column>
      <LabelColumn>false</LabelColumn>
      <Id>1</Id>
      <Labels>
        <Label Id="1" Label="6 Months Ended" />
        <Label Id="2" Label="Jun. 30, 2009" />
        <Label Id="4" Label="USD / shares" />
      </Labels>
      <CurrencySymbol>$</CurrencySymbol>
      <hasSegments>false</hasSegments>
      <hasScenarios>false</hasScenarios>
      <Segments />
      <Scenarios />
      <Units>
        <Unit>
          <UnitID>USD</UnitID>
          <UnitType>Standard</UnitType>
          <StandardMeasure>
            <MeasureSchema>http://www.xbrl.org/2003/iso4217</MeasureSchema>
            <MeasureValue>USD</MeasureValue>
            <MeasureNamespace>iso4217</MeasureNamespace>
          </StandardMeasure>
          <Scale>0</Scale>
        </Unit>
        <Unit>
          <UnitID>USDEPS</UnitID>
          <UnitType>Divide</UnitType>
          <NumeratorMeasure>
            <MeasureSchema>http://www.xbrl.org/2003/iso4217</MeasureSchema>
            <MeasureValue>USD</MeasureValue>
            <MeasureNamespace>iso4217</MeasureNamespace>
          </NumeratorMeasure>
          <DenominatorMeasure>
            <MeasureSchema>http://www.xbrl.org/2003/instance</MeasureSchema>
            <MeasureValue>shares</MeasureValue>
            <MeasureNamespace>xbrli</MeasureNamespace>
          </DenominatorMeasure>
          <Scale>0</Scale>
        </Unit>
      </Units>
    </Column>
  </Columns>
  <Rows>
    <Row>
      <Id>2</Id>
      <Label>Employee Separation Costs [Abstract]</Label>
      <Level>0</Level>
      <ElementName>bni_EmployeeSeparationCostsAbstract</ElementName>
      <ElementPrefix>bni</ElementPrefix>
      <IsBaseElement>false</IsBaseElement>
      <BalanceType>na</BalanceType>
      <PeriodType>duration</PeriodType>
      <ElementDataType>string</ElementDataType>
      <ShortDefinition>Employee Separation Costs.</ShortDefinition>
      <IsReportTitle>false</IsReportTitle>
      <IsSegmentTitle>false</IsSegmentTitle>
      <IsSubReportEnd>false</IsSubReportEnd>
      <IsCalendarTitle>false</IsCalendarTitle>
      <IsTuple>false</IsTuple>
      <IsAbstractGroupTitle>true</IsAbstractGroupTitle>
      <IsBeginningBalance>false</IsBeginningBalance>
      <IsEndingBalance>false</IsEndingBalance>
      <IsEPS>false</IsEPS>
      <Cells>
        <Cell>
          <Id>1</Id>
          <ShowCurrencySymbol>false</ShowCurrencySymbol>
          <IsNumeric>false</IsNumeric>
          <NumericAmount>0</NumericAmount>
          <RoundedNumericAmount>0</RoundedNumericAmount>
          <NonNumbericText />
          <NonNumericTextHeader />
          <FootnoteIndexer />
          <hasSegments>false</hasSegments>
          <hasScenarios>false</hasScenarios>
        </Cell>
      </Cells>
      <ElementDefenition>Employee Separation Costs.</ElementDefenition>
      <IsTotalLabel>false</IsTotalLabel>
    </Row>
    <Row>
      <Id>3</Id>
      <Label>Employee Separation Costs</Label>
      <Level>1</Level>
      <ElementName>bni_EmployeeSeparationCostsTextBlock</ElementName>
      <ElementPrefix>bni</ElementPrefix>
      <IsBaseElement>false</IsBaseElement>
      <BalanceType>na</BalanceType>
      <PeriodType>duration</PeriodType>
      <ElementDataType>string</ElementDataType>
      <ShortDefinition>Disclosure of liabilities representing deferred benefits payable upon separation or retirement of certain active employees...</ShortDefinition>
      <IsReportTitle>false</IsReportTitle>
      <IsSegmentTitle>false</IsSegmentTitle>
      <IsSubReportEnd>false</IsSubReportEnd>
      <IsCalendarTitle>false</IsCalendarTitle>
      <IsTuple>false</IsTuple>
      <IsAbstractGroupTitle>false</IsAbstractGroupTitle>
      <IsBeginningBalance>false</IsBeginningBalance>
      <IsEndingBalance>false</IsEndingBalance>
      <IsEPS>false</IsEPS>
      <Cells>
        <Cell>
          <Id>1</Id>
          <ShowCurrencySymbol>false</ShowCurrencySymbol>
          <IsNumeric>false</IsNumeric>
          <NumericAmount>0</NumericAmount>
          <RoundedNumericAmount>0</RoundedNumericAmount>
          <NonNumbericText>&lt;!--DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd" --&gt;
   &lt;html&gt;
   &lt;head&gt;&lt;/head&gt;
   &lt;body&gt;
   &lt;!-- Begin Block Tagged Note 6 - bni:EmployeeSeparationCostsTextBlock--&gt;
   &lt;div style="font-family: 'Times New Roman',Times,serif"&gt;
   &lt;div align="left" style="font-size: 10pt; margin-top: 12pt"&gt;&lt;b&gt;6. Employee Separation Costs&lt;/b&gt;
   &lt;/div&gt;
   &lt;div align="left" style="font-size: 10pt; margin-top: 6pt"&gt;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;Employee separation costs activity was as follows (in millions):
   &lt;/div&gt;
   &lt;div align="center"&gt;
   &lt;table style="font-size: 10pt" cellspacing="0" border="0" cellpadding="0" width="100%"&gt;
   &lt;!-- Begin Table Head --&gt;
   &lt;tr valign="bottom"&gt;
       &lt;td width="76%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="5%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="1%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="5%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="1%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="5%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="1%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="5%"&gt;&amp;#160;&lt;/td&gt;
       &lt;td width="1%"&gt;&amp;#160;&lt;/td&gt;
   &lt;/tr&gt;
   &lt;tr style="font-size: 8pt" valign="bottom"&gt;
       &lt;td nowrap="nowrap" align="left" style="border-bottom: 1px solid #000000"&gt;&lt;b&gt;Six Months Ended June 30,&lt;/b&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td nowrap="nowrap" align="center" colspan="2" style="border-bottom: 1px solid #000000"&gt;&lt;b&gt;2009&lt;/b&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td nowrap="nowrap" align="center" colspan="2" style="border-bottom: 1px solid #000000"&gt;&lt;b&gt;2008&lt;/b&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
   &lt;/tr&gt;
   &lt;!-- End Table Head --&gt;
   &lt;!-- Begin Table Body --&gt;
   &lt;tr valign="bottom" style="background: #cceeff"&gt;
       &lt;td&gt;
   &lt;div style="margin-left:15px; text-indent:-15px"&gt;Beginning balance at January&amp;#160;1,
   &lt;/div&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="left"&gt;$&lt;/td&gt;
       &lt;td align="right"&gt;79&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="left"&gt;$&lt;/td&gt;
       &lt;td align="right"&gt;91&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
   &lt;/tr&gt;
   &lt;tr valign="bottom"&gt;
       &lt;td&gt;
   &lt;div style="margin-left:15px; text-indent:-15px"&gt;Accruals
   &lt;/div&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="right"&gt;9&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="right"&gt;3&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
   &lt;/tr&gt;
   &lt;tr valign="bottom" style="background: #cceeff"&gt;
       &lt;td&gt;
   &lt;div style="margin-left:15px; text-indent:-15px"&gt;Payments
   &lt;/div&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td nowrap="nowrap" align="left"&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="right"&gt;(6&lt;/td&gt;
       &lt;td nowrap="nowrap"&gt;)&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td nowrap="nowrap" align="left"&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="right"&gt;(7&lt;/td&gt;
       &lt;td nowrap="nowrap"&gt;)&lt;/td&gt;
   &lt;/tr&gt;
   &lt;tr style="font-size: 1px"&gt;
       &lt;td&gt;
   &lt;div style="margin-left:15px; text-indent:-15px"&gt;&amp;#160;
   &lt;/div&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td nowrap="nowrap" colspan="2" align="right" style="border-top: 1px solid #000000"&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td nowrap="nowrap" colspan="2" align="right" style="border-top: 1px solid #000000"&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
   &lt;/tr&gt;
   &lt;tr valign="bottom"&gt;
       &lt;td&gt;
   &lt;div style="margin-left:30px; text-indent:-15px"&gt;Ending balance at June&amp;#160;30,
   &lt;/div&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="left"&gt;$&lt;/td&gt;
       &lt;td align="right"&gt;82&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td align="left"&gt;$&lt;/td&gt;
       &lt;td align="right"&gt;87&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
   &lt;/tr&gt;
   &lt;tr style="font-size: 1px"&gt;
       &lt;td&gt;
   &lt;div style="margin-left:15px; text-indent:-15px"&gt;&amp;#160;
   &lt;/div&gt;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
           &lt;td nowrap="nowrap" colspan="2" align="right" style="border-top: 3px double #000000"&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
           &lt;td nowrap="nowrap" colspan="2" align="right" style="border-top: 3px double #000000"&gt;&amp;#160;&lt;/td&gt;
       &lt;td&gt;&amp;#160;&lt;/td&gt;
   &lt;/tr&gt;
   &lt;!-- End Table Body --&gt;
   &lt;/table&gt;
   &lt;/div&gt;
   &lt;div align="left" style="font-size: 10pt; margin-top: 6pt"&gt;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;Employee separation liabilities of $82&amp;#160;million were included in the Consolidated Balance Sheet
       at June&amp;#160;30, 2009, and principally represent the following: (i) $80&amp;#160;million for deferred benefits
       payable upon separation or retirement to certain active conductors, trainmen and locomotive
       engineers; (ii)&amp;#160;less than $1&amp;#160;million for employee-related severance costs for the consolidation of
       clerical functions, material handlers in mechanical shops and trainmen on reserve boards; and (iii)
   $2&amp;#160;million for certain non-union employee severance costs. Employee separation expenses are
       recorded in materials and other in the Consolidated Statements of Income. At June&amp;#160;30, 2009, $17
   million of the remaining liabilities was included in current liabilities.
   &lt;/div&gt;
   &lt;div align="left" style="font-size: 10pt; margin-top: 6pt"&gt;&amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;The deferred benefits payable upon separation or retirement to certain active conductors,
       trainmen and locomotive engineers were primarily incurred in connection with labor agreements
       reached prior to the business combination of BNSF&amp;#8217;s predecessor companies, Burlington Northern Inc.
   and Santa Fe Pacific Corporation. These agreements, among other things, reduced train crew sizes
       and allowed for more flexible work rules. The majority of the remaining costs will be paid between
       2009 and 2020. As of June&amp;#160;30, 2009, the Company had updated its estimate and recorded an additional
       liability of $9&amp;#160;million related to deferred benefits. The remaining costs for (ii)&amp;#160;above are
       expected to be paid out between 2009 and approximately 2011, and the costs for (iii)&amp;#160;above are
       expected be paid out between 2009 and 2021 based on deferral elections made by the affected
       employees.
   &lt;/div&gt;
   &lt;/div&gt;
   &lt;/body&gt;
   &lt;/html&gt;
</NonNumbericText>
          <NonNumericTextHeader>&lt;!-- Begin Block Tagged Note 6 - bni:EmployeeSeparationCostsTextBlock--&gt;

   6. Employee Separation Costs

   &amp;#160;&amp;#160;&amp;#160;&amp;#160;&amp;#160;Employee</NonNumericTextHeader>
          <FootnoteIndexer />
          <hasSegments>false</hasSegments>
          <hasScenarios>false</hasScenarios>
        </Cell>
      </Cells>
      <ElementDefenition>Disclosure of liabilities representing deferred benefits payable upon separation or retirement of certain active employees incurred in connection with labor agreements reached prior to a business combination and severance costs for certain union and non union employees.</ElementDefenition>
      <ElementReferences>No authoritative reference available.</ElementReferences>
      <IsTotalLabel>false</IsTotalLabel>
    </Row>
  </Rows>
  <Footnotes />
  <ComparabilityReport>false</ComparabilityReport>
  <NumberOfCols>1</NumberOfCols>
  <NumberOfRows>2</NumberOfRows>
  <HasScenarios>false</HasScenarios>
  <MonetaryRoundingLevel>UnKnown</MonetaryRoundingLevel>
  <SharesRoundingLevel>UnKnown</SharesRoundingLevel>
  <PerShareRoundingLevel>UnKnown</PerShareRoundingLevel>
  <HasPureData>false</HasPureData>
  <SharesShouldBeRounded>true</SharesShouldBeRounded>
</InstanceReport>
