This is my user control code that's working:
<%@ Control Language="C#" AutoEventWireup="true" CodeBehind="test.ascx.cs" Inherits="SchmitsList.controls.test" %>
<%@ Register Assembly="AjaxControlToolkit" Namespace="AjaxControlToolkit" TagPrefix="cc21" %>
<table>
<tr valign="middle">
<td>
<asp:TextBox ID="multiHandle2_1_BoundControl" runat="server" Width="30" Text="25" />
</td>
<td valign="top">
<asp:TextBox ID="sliderTwo" runat="server" Style="display: none;" />
<cc21:MultiHandleSliderExtender ID="multiHandleSliderExtenderTwo" runat="server"
BehaviorID="multiHandleSliderExtenderTwo" TargetControlID="sliderTwo" Minimum="0"
Maximum="100" Length="175" TooltipText="{0}" Orientation="Vertical" EnableHandleAnimation="true"
EnableKeyboard="false" EnableMouseWheel="false" ShowHandleDragStyle="true" ShowHandleHoverStyle="true">
<MultiHandleSliderTargets>
<cc21:MultiHandleSliderTarget ControlID="multiHandle2_1_BoundControl" />
<cc21:MultiHandleSliderTarget ControlID="multiHandle2_2_BoundControl" />
</MultiHandleSliderTargets>
</cc21:MultiHandleSliderExtender>
</td>
<td style="padding-left: 21px">
<asp:TextBox ID="multiHandle2_2_BoundControl" runat="server" Width="30" Text="75" />
</td>
</tr>
</table>
Note the second line where I registert the toolkit.