The ability to isolate, characterize, and expand distinct tumor cell populations from primary tissue or xenografts is vital to identifying molecular mechanisms specific to cancer stem cells. Once cells have been extracted from tissue, there are multiple methods by which they can be sorted and cultured. We will describe the approaches that can be taken from cancer stem cell isolation through expansion, including Magnetic-activated Cell Sorting (MACS), Fluorescence-activated Cell Sorting (FACS), the use of reporter systems, and various cell culture methods.