For anyone using Windows Server 2003 (like me) you will likely run into this issue sooner or later. Thankfully, for me it was later since my Windows box is a spare. Ironically, it's for working out kinks like this!

You'll probably find this useful blog post at the top of your SERPs, but I'll pass it along anyway just in case:

FLV 404 Error On Windows 2003 Server
Posted July 26, 2007 at 11:48 AM
http://www.bennadel.com/blog/866-FLV-404-Error-On-Windows-2003-Server.htm

Thanks to Ben Nadel!