It is rather easy to expand ex vivo keratinocytes and their stem cells from many species, such as human being, monkey, rabbit, with the exception of mouse. From the double transgenic mice expressing SV40 Tag driven by the tetracycline-transactivator (tTA, tet-off), a mouse keratinocytic stem cell line has been established and partially characterized. Unlike primary keratinocytes of other species, differentiation of these keratinocytic stem cells does not occur automatically. It requires either elements provided by built-in cytokines, the subcutaneous microenvironment of SCID-NOD mice, or co-culturing with a feeder cell line. Applying such approaches, the derived cell clones have been isolated and partially characterized. A model is proposed to suggest the lineage development of mouse keratinocytic stem cells. Such doxycycline-regulated, stage-specific keratinocytic cell clones may provide a useful source of cells for keratinocyte functional genomic analysis and drug screening.