In vitro cell culture is a necessary prerequisite in acquiring a thorough understanding of the biology and behavior of the cells of interest and is a critical first step in developing cellular therapies. Somatic stem cell biology is concerned with stem cells of the adult and how they may be utilized in regenerating tissue and ameliorating disease. Moreover, the incidence of disease increases with age, hence the demand for therapeutics is greatest among mature individuals. Therefore, an ability to grow and manipulate primary adult epithelial keratinocytes in vitro is of paramount importance in gaining insights into the biology of skin that may have clinical implications. A methodology has been developed that will enable investigators to isolate and serially culture adult basal keratinocytes from the epidermis of the mouse in a supplemented culture medium that selects for epithelial lineages and enhances their proliferation while inhibiting differentiation through many passages.