Generate FLV video with alpha channel using open source tools

AviDemux, open first Image via "File"->"Open", add following images via "File"->"Append...".

Choose "Sorenson Spark" in Video, "FLV" in Format. Configure your settings, play with filter, then Save as Video, and you're done!

In a browser, you can display an FLV with alpha channel with just changing some parameters.
See the following article for an example: Alpha Video and HTML: How did they do that?

If you are looking for software to do that, I don't know of any good open-source solution, but there is a low-budget commercial alternative : Moyea Flash Video MX Pro ($99.95), with a free trial for checking it out. You can watch a demo video tutorial here.

EDIT

The K-Lite Codec Pack is said in the Codec Guide to contain all the flavors of the VP6 codec : VP60, VP61, VP62, VP6A, VP6F.