Objective: To differentiate human embryonic stem cells (hESCs) into keratinocytes (K-hESCs) and analyse the expression characteristics of biomarkers of K-hESCs. Methods: The hESCs of line H9 were seeded on matrigel in mTeSR1 medium. The hESCs were directly differentiated into keratinocytes in epithelial differentiation medium with bone morphogenetic protein 4, retinoic acid and N2 supplement. The karyotype of K-hESCs was analyzed, comparing the gene expression differences of K-hESCs with human gingival epithelial cells (HGECs), human immortalized oral epithelial cells (HIOECs) and HaCaT by Realtime PCR. Molecular characteristics of the cell differentiation were observed throughout the process by immunocytochemical techniques. Results: H9hESCs were successfully differentiated into the cells that exhibited characteristics of keratinocytes in epithelial differentiation medium. The karyotype of K-hESCs was 46, XX; and the keratinocyte gene p63 expression in K-hESCs was significantly lower than that in HaCaT (P<0.05), but there was no significant difference of p63 expression in K-hESCs, comparing with that in HGECs and HIOECs (P>0.05). Conclusion: H9-hESCs could be directly differentiated into K-hESCs. The gene expression of K-hESCs was similar to that of epithelial cells in the early stage of monolayer cells differentiation with high proliferative activity.