Primary keratinocytes are harvested from the dorsal skin of 7-week-old mice. Euthanized mice are clipped and cleaned with serial washes in povidone iodine and ethanol solutions. The dorsal skin is removed and treated with a mild 32 C trypsinization to detach the dermis from the epidermis. Keratinocytes harvested by this method can be used for molecular biology, biochemistry, or numerous ex vivo procedures relevant to cancer research such as clonal culture or fluorescence-activated cell sorting (FACS). FACS, a type of flow cytometry, is a quantitative means of sorting cell mixtures using diameter, fluorescent dye, and the molecular charge of each cell type in the sample suspension. This technique is performed using specialized shared instruments at The Hormel Institute, including the BD FACSAria II for cell sorting and the BD FACSCalibur for analysis only. The equipment and techniques presented here are useful as an ex vivo readout of in vivo experiments on skin and for preparation of enriched stem cells for further study in molecular biology and other applications.