This study explores the effects of enhancing the definitive hematopoiesis(DH)signals during the differentiation of human embryonic stem cells(hESCs)into hematopoietic stem/progenitor cells on the generation efficiency and effector function of natural killer(NK)cells generated from hESCs(also known as hESC-NK cells).The hESC(H1)were transformed into embryoid bodies(Ebs)by centrifugation,and during the induction of K562,were used to analyze the efficiency of hematopoietic differentiation,the efficiency of NK cell generation from hESC,the in vitro effector functions,and the expression of effector function related surface receptors.Compared to the control group,the DH group had a significant increase in the number of arterial hematopoietic endothelial cells(CD34+DLL4+)and a significant decrease in primitive hematopoietic related cells(CD34-CD43+)on day 8 of hematopoietic differentiation(P<0.05).On day 28 of NK cell differentiation,the DH group demonstrated a significant increase in the number of NK cells(CD45+CD56+),while a slight increase in the expression of effector function-related molecules such as IFN-γ,Granzyme B,Perforin and CD107a without statistical significance.Furthermore,the activation receptors CD16a and CD69 were significantly increased,NKP46 was significantly decreased,the inhibitory receptor NKG2A was significantly increased,while CD96 was significantly decreased on hESC-NK cells of DH groups(P<0.05).Conclusively,enhancing the signals for definitive hematopoiesis during hESC differentiation into hematopoietic stem/progenitor cells significantly improves the yield of NK cells and the expression of CD16a without affecting their in vitro effector functions.Our study provides a new approach to improving the efficiency of hESC-NK cell or iPSC-NK cell generation.