This unit describes protocols for working with colon cancer stem cells. To work with these cells one must start by generating single-cell suspensions from human colon cancer tissue. These cell suspensions are sorted using flow cytometry-assisted cell sorting to fractionate the cells into tumor-initiating and nontumor-initiating subsets. Once the cells have been fractionated, they must be functionally tested to determine tumor-forming capacity, the gold standard being the in vivo xenograft assay. Methods have also been developed to grow these cells in vitro in a sphere-forming assay. This unit will describe how to isolate and functionally test colon cancer stem cells, as well as provide advice on the potential challenges of the research.