The cancer stem cell hypothesis states that within a tumor only a subset of cells, the “cancer stem cells” (CSC), are capable of initiating and propagating the disease. In various cancers such cells have been identified and prospectively isolated based on the presence of specific cell surface antigens. In head and neck squamous cell carcinomas, we have shown that the CSCs are contained within the CD44+ subset of tumor cells. This subset contains cells capable of initiating tumor growth in mice that recapitulates the original tumor heterogeneity. Furthermore, they have a primitive cellular morphology, express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. The methods used to purify this subset of tumorigenic cells, to characterize their gene expression profiles, and to identify their physical location within the context of the whole tumor are described here.