The basal layer of human interfollicular epidermis is thought to contain a minor compartment of quiescent or slowly cycling epithelial stem cells. These primitive keratinocytes give rise to the progenitors, which are the proliferating keratinocytes and which can be defined as early to late progenitors, according to their differentiation status. Because of the intrinsic heterogeneity of the basal layer, the development of new methods suitable for functional analysis of basal keratinocytes directly isolated from skin samples is greatly needed. We describe here a new method that allows a rapid and multiparallel deposition of single keratinocytes into 96-well plates, using flow cytometry. The first step of the process allows the clonal analysis of the growth potential of freshly isolated epithelial cells in primary cultures. In a second step, various techniques of functional characterization can be performed on the progeny of the cloned cell, including the generation of reconstructed epidermis, colony assays, and secondary cloning. In a third step, a long-term characterization of the progeny of the cloned keratinocytes can be performed, either by successive subclonings or mass expansion cultures.