The study of adult stem cell populations provides insight into the mechanisms that regulate tissue maintenance in normal physiology and many disease states. With an impressive rate of epithelial renewal driven by a pool of multipotent stem cells, the intestine is a particularly advantageous model system for the study of adult stem cells. Until recently, the isolation and in vitro study of intestinal epithelial stem cells (IESCs) was not possible due to the lack of biomarkers and culture techniques. However, advances in molecular characterization and culture of IESCs have made in vitro studies on this cell type amenable to most laboratories. The methods described in this chapter will allow the investigator to adapt newly established techniques toward downstream analysis of IESCs in vitro.