Objective Insulin-producing β cells derived from human pluripotent stem cells including hESCs and iPSCs could not only be one of the prominent therapy of type 1 diabetes but also suggest in vitro disease model of genetic disease. In this regard, human embryonic stem cells are differentiated into pancreatic endocrine cells through defined spatiotemporal regulation. Methods 1) Q-RT PCR; 2) Immunofluorescence analysis Results 1) For definitive endoderm stage induction, both Activin/Nodal signaling and WNT signaling are activated. Human embryonic stem cells were differentiated more efficiently and rapidly when WNT signaling was activated with Activin/Nodal signaling compared to Actin/Nodal signaling activation alone. 2) Single cell dissociation and replating after definitive endoderm stage reduced hepatic differentiation process significantly. This mechanical modulation in the replating condition increased the expression of mesenchymal cell type markers such as N-Cadherin, Fibronectin in more homogeneous manner. However, epithelial cell type marker gene expression was not changed after replating. Conclusion 1) The first stage of in vitro differentiation from hESCs is definitive endoderm. To induce the appropriate definitive endoderm state for pancreatic specification, WNT signaling was activated with Activn/Nodal signaling showing more rapid differentiation. This indicates the critical point of the state of definitive endoderm stage for pancreatic cell fate specification. 2) After definitive endoderm stage, single cell dissociation and replating process significantly decreased the expression of AFP, one of the hepatic cell type markers. In addition, the expression of mesenchymal cell markers such as N-Cadherin, Fibronectin, Vimentin was homogeneously increased after replating. This phenomenon indicates micro-environmental alteration enhances EMT during pancreatic specification by recapitulating in vivo development. As a result, not only could the spontaneous hepatic cell fate differentiation be inhibited but also pancreatic specification could be more efficiently regulated. 3) For more efficient endocrine differentiation and insulin-producing β cell maturation, defined signaling modulation is required after pancreatic cell fate specification.