I'd recommend keeping the template intact and drawing on a blank layer on top of it. Then, once you're ready to use / test the tileset, you can hide the template layer and your tiles should automatically have the transparency around them.
Remember - for a tileset to be usable, every tile (except the blank tile in the top left corner) must contain SOMETHING. If any tile is left blank / transparent, the KS Editor will not let you use the tileset until it's filled with something. This is why you see the letters B, L, A, N and K on some of the public (default) tilesets. Usually, I make a second copy of the template and just cut out the squares I'm using, thus: