that depends i guess, what tools are you using to create them?
if you're using iconbuilder pro with adobe photoshop, just make sure that you specify a transparent background when you create the new file. when you import your layer into iconbuilder pro, the background will be transparent.
if you're using the tool supplied by apple's developer tools, you basically just want to drag transparen tiffs to the wells. here, when you save your tiff in photoshop, make sure you use lyz.