Human embryonic stem cells (hESCs)-derived keratinocytes hold great clinical and research potential. However, the current techniques are hampered by the use of xenogenic components that limits their clinical application. Here we demonstrated an efficient differentiation of H9 hESCs (H9-hESCs) into keratinocytes (H9-Kert) with the minimum use of animal-derived materials. For differentiation, we established two microenvironment systems originated from H9-hESCs (autogenic microenvironment). These autogenic microenvironment systems consist of an autogenic coculture system (ACC) and an autogenic feeder-free system (AFF). In addition, we showed a stage-specific effect of Activin in promoting keratinocyte differentiation from H9-hESCs while repressing the expression of early neural markers in the ACC system. Furthermore, we also explained the effect of Activin in construction of the AFF system made up of extracellular matrix similar to basement membrane extracted from H9-hESC-derived fibroblasts. H9-Kert differentiated in both systems expressed keratinocyte markers at mRNA and protein levels. H9-Kert were also able to undergo terminal differentiation in high Ca(2+) medium. These findings support the transition toward the establishment of an animal-free microenvironment for successful differentiation of hESCs into keratinocytes for potential clinical application.