Induced pluripotent stem cells (iPSCs) have great potential due to their proliferative ability, and their potential to differentiate into different cell lineages for disease modeling and therapeutical applications. However, realization of iPSC potential depends on the differentiation of iPSCs into different desired cell lineages. We succeeded in generating different cell types, including keratinocytes (KC), for the purpose of constructing human skin for disease modeling and clinical applications. Here, we investigated the molecular alterations during iPSC differentiation into KC. We reprogrammed PBMCs or fibroblasts into iPSCs with non-integrating systems (episomal vectors or Sendai virus). Established iPSCs expressed pluripotency markers and exhibited trilineage differentiation. We then used administration of 1 μM retinoic acid and 10 ng/μl BMP4 for 6 days for KC differentiation from iPSCs. The differentiating cells were maintained for 60 days for maturation before subculturing. RNA was extracted at different time points (day7, day14, day21, day30, day45, and day60) for RNA-seq analysis. Analysis of the RNA-seq data showed that there were around 7000 genes differentially expressed (using p>0.01 as cutoffs) between normal primary KC and the cells 7 days after differentiation initiation. As the maturation progressed, the number of differentially expressed genes decreased gradually to 4000 genes (at day30) compared with primary KC. Interestingly, the differences in the number of differentially expressed genes between different time points were less than 2000 (again, using p>0.01 as cutoffs). Many pathways, such as TGFβ and PI3K, were detected during KC differentiation/maturation. With the guidance from RNA-seq data, we are perturbing different signaling pathways to improve KC differentiation efficiency from iPSCs. Efficient iPSC/KC differentiation will allow us to obtain large amount of high quality KC from iPSCs for disease modeling and/or clinical applications.