This protocol describes a technically simple in vivo assay of long-term skin regeneration in human skin, providing a reliable method for epidermal tissue reconstitution using small numbers of several types of epithelial cells, from epithelial cell lines to primary epithelial stem cells and transplanted with or without prior culture. The model relies on the repopulation of devitalized rat tracheas by human keratinocyte suspensions following subcutaneous transplantation into immunodeficient mice. Here, we describe complete optimization and characterization of this model for robust regeneration of epithelium from cell suspensions of a limited number of primary human keratinocytes.