Recent work from our laboratory has led to the development and validation of fluorescence-activated cell sorting (FACS)-based techniques to prospectively isolate viable keratinocyte stem cells from both human and murine skin. Here we describe a step-by-step method to apply our technique to isolate epidermal keratinocytes from skin tissue, process them for immunofluorescent staining for cell surface markers, and subject them to fluorescence-activated cell sorting to obtain the stem, transient amplifying, and early differentiating keratinocyte fractions. These viable cells can then be placed into culture for further analysis or directly into keratinocyte assays, such as organotypic cultures or in vivo transplantation. This method will be useful for the complete biological characterization of keratinocyte progenitors with respect to wound healing, carcinogenesis, and therapeutic manipulation.