If you want different "I spy" blocks, you would probably need to use regular cotton for the I spy blocks. I have seen some quilts that used flannel as the sashing with cottons. You could also use the regular fabrics for the front and back it in flannel. Whatever your bent is for pre-washing fabrics, always pre-wash flannel. It shrinks more and at different rates than the regular cottons.
I have never used fleece on the backs of my quilts. I hear that it stretches a lot.
As far as the number of squares, determine the size you want the quilt to be, if you plan on using sashing between the blocks, if you want to add borders, etc. When you are figuring sizes remember that a 5" block will really sew up as 4 1/2" any sashing or borders, take a 1/2" off each for seams when figuring sizes. I hope this helps a little!