Today, I had a Windows Server 2003 system, on which I freshly installed .NET 4.0 and wanted to deploy an ASP.NET 4.0 application in it. Sounds simple enough, but everytime I tried to access the application, I got a 404 error. In my case, I had an MVC2 application, for which I also enabled the wildcard mapping (I know, it's not required with 4.0, but I did it anyways, just in case). When it was enabled, I got 404s for every request. When I took it out, at least a simple text file was delivered instead of a 404 error.
Well, the solution was very simple, but it took a while to find. The ASP.NET 4.0 web service extension was disabled. So, if you keep getting 404s for your ASP.NET application, check in the Webservice Extensions folder in the IIS Manager if the ASP.NET 4.0 extension is enabled.