For many years, cell therapies have been hampered by limited availability and inter-batch variability of primary cells. Human embryonic stem cell (hESC) can give rise to specialized cells like keratinocytes and recently emerged as a virtually unlimited source of potential therapeutic cells. However, xenogeneic components in differentiation cocktails have been limiting the clinical potential of hESC-derived keratinocytes (hESCs-Kert). Here, we demonstrated efficient differentiation of H9 human embryonic stem cells (H9-hESCs) into keratinocytes (H9-Kert(ACC)) in an autogenic co-culture system. We used activin as the main factor to induce keratinocyte differentiation. H9-Kert(ACC) expressed keratinocyte markers at mRNA and protein levels. Establishment of such animal-free microenvironment for keratinocyte differentiation will accelerate potential clinical application of hESCs.