The p63 transcription factor is identified as a marker for keratinocyte stem cells in human corneal and epidermal tissues. This study shows that p63 is specifically expressed by stem cells and not by transient amplifying (TA) cells. In the cornea, p63 is found in the basal layer of the limbal epithelium but not in TA cells covering the corneal surface. In culture, keratinocyte stem cells generate holoclones, while TA cells generate paraclones. Clonal analysis revealed that p63 is abundantly expressed in holoclones but undetectable in paraclones. TA keratinocytes, after leaving the stem cell compartment (meroclones), show reduced p63 levels despite maintaining high proliferative capacity. The sigma isoform of the 14-3-3 family of proteins promotes clonal evolution, and down-regulating 14-3-3σ in keratinocytes maintains them in the stem cell compartment and preserves p63 expression. These findings confirm that p63 is a specific marker for keratinocyte stem cells, which has implications for epithelial cell therapy and tumorigenesis research. The study also highlights the importance of p63 in maintaining stem cell identity and its potential role in epithelial regeneration and cancer development.The p63 transcription factor is identified as a marker for keratinocyte stem cells in human corneal and epidermal tissues. This study shows that p63 is specifically expressed by stem cells and not by transient amplifying (TA) cells. In the cornea, p63 is found in the basal layer of the limbal epithelium but not in TA cells covering the corneal surface. In culture, keratinocyte stem cells generate holoclones, while TA cells generate paraclones. Clonal analysis revealed that p63 is abundantly expressed in holoclones but undetectable in paraclones. TA keratinocytes, after leaving the stem cell compartment (meroclones), show reduced p63 levels despite maintaining high proliferative capacity. The sigma isoform of the 14-3-3 family of proteins promotes clonal evolution, and down-regulating 14-3-3σ in keratinocytes maintains them in the stem cell compartment and preserves p63 expression. These findings confirm that p63 is a specific marker for keratinocyte stem cells, which has implications for epithelial cell therapy and tumorigenesis research. The study also highlights the importance of p63 in maintaining stem cell identity and its potential role in epithelial regeneration and cancer development.