Background: Inherent biological properties of human em-bryonic stem cells (hESCs) including differentiation into any somatic cell type and self-renewing have opened up numer-ous opportunities in biomedical research and cell replacement therapies. Under suitable culture conditions, hESCs can be sta-bilized. However, these cells have the ability to sense and re-sponse to various environmental signals. Extrinsic fluctuation in their environment could direct cell death or differentiation in hESCs. P38 mitogen activated protein kinases (p38-MAPK) is a signaling pathway that is activated with a variety of environ-mental stress and plays a role in cell differentiation, apoptosis, and autophagy. Nevertheless, P38-MAPK function on hESCs is remained unclear.Materials and Methods: Here, we suppressed p38-MAPK in different hESC lines (Royan H5 and Royan H6) by using a small molecule inhibitory, SB203580 (10 µm), in continuous passages. After a few passages a series of stemness features were evaluated. The results were compared with non-treated hESCs as a control group.Results: We showed that p38-MAPK inhibition resulted in changing morphology of hESCs from flat form to small and dome-shaped. These cells displayed a high growth rate, in-creased clonogenicity, and were resistance to single cell dis-sociation by trypsin relative to non-tread hESC. Furthermore, we found that some naïve specific genes were upregulated in treated-hESC with p38-MAPK inhibitor such as; NANOG, STELLA, KLF2, KLF5, TFCP2L1, and DNMT3L. These cells simultaneously expressed pluripotency and lineage specific markers. Spontaneous differentiation showed that P38i-treated cells efficiently expressed AFP and ALB genes which are rep-resentative for hepatocyte differentiation. In addition, we de-tected higher percentage of endodermal like cells in teratoma formed by p38i-treated hESCs. We also found more than two fold increase in SOX17 expression at protein level when p38i-treated cells were directly differentiated into endoderm in com-parison with non-tread hESCs.Conclusion: Taken together, our results suggest that suppres-sion of p38-MAPK in hESCs could provide a cell resource for robust endoderm cells in order to facilitate in mature and reli-able hepatocyte and pancreases cell differentiation.