Here's an interesting one from Jeff Berkowitz and Phil Hochstetler:
Phil has discovered that MS hotfix 887219 / MS05-004 (http://www.microsoft.com/technet/security/Bulletin/MS05-004.mspx), released last Wednesday, completely breaks our site. At least on one test machine--happily we caught it before it reached Production. With the hotfix installed, all our virtual roots (three of them, one containing a web application and two containing collections of web service pages) return 404s exclusively. Backing out just this one hotfix solves the problem.
The fix was found by Phil on Google Groups: Go to IIS Admin, open Properties on Web Site, Home directory tab, Local Path text box: make sure there is no trailing slash.