ID1 can mediate transforming growth factor-β (TGF-β)/activin receptor-like kinase-1 (ALK1)-induced (and Smad-dependent) migration in endothelial cells (ECs). However, the role that ID1 plays during differentiation of human embryonic stem cells (hESCs) into ECs induced by TGF-β1 remains unclear. In this study, a hESC differentiation model that recapitulates the developmental steps of vasculogenesis during the early stages of embryonic development was used to explore this question. We found that TGF-β1 increases endothelial cell differentiation and inhibits endothelial tube formation. Furthermore, at an early stage of differentiation, TGF-β1 may induce in vitro differentiation of hESCs into ECs by inhibiting expression of ID1, while at a later stage of differentiation, TGF-β1 may stimulate the proliferation and migration of ECs via the ALK1/Smad1/5/ID1 pathway. Downregulation of ID1 by gene silencing can lead to acceleration of TGF-β1-induced hESC differentiation into ECs and inhibition of proliferation and migration of ECs. This study may reveal some mechanisms of in vivo vasculogenesis in the early stages of embryonic development.