Intercellular adhesion is crucial in regulating stemness maintenance and differentiation initiation of embryonic stem cells (ESCs), which is also cooperated with extracellular mechanical microenvironment. Here an in vitro model was used to elucidate the effects of E-cadherin complexes on definitive endoderm (DE)-directed differentiation of hESCs (H1 cells), when the cells were seeded on polyacrylamide hydrogels with varied stiffnesses. Results indicated that stiff substrate increased the proportion of H1 cells differentiating into DE cells and intercellular E-cadherin expression was reduced with progressive stages at same stiffness, presenting a negative correlation of E-cadherin expression with differentiating progress or substrate stiffness. Blocking E-cadherin enhanced the productivity of differentiated cells and promoted the disassembly of intercellular adhesions by translocating YAP into nuclei, which was positively correlated with GATA6 and CXCR4 expressions in a stiffness-dependent manner. This work provided an insight into understanding the roles of E-cadherin-related intercellular adhesion and substrate stiffness in DE-directed differentiation of hESCs.