The transition from hemogenic endothelial cells (HECs) to hematopoietic stem/progenitor cells (HS/PCs), or endothelial to hematopoietic transition (EHT), is a critical step during hematopoiesis. However, little is known about the molecular determinants of HECs due to the challenge in defining HECs. We report here the generation of GATA2w/eGFP reporter in human embryonic stem cells (hESCs) to mark cells expressing GATA2, a critical gene for EHT. We show that during differentiation, functional HECs are almost exclusively GATA2/eGFP+. We then constructed a regulatory network for HEC determination and also identified a panel of positive or negative surface markers for discriminating HECs from non-hemogenic ECs. Among them, ITGB3 (CD61) precisely labeled HECs both in hESC differentiation and embryonic day 10 mouse embryos. These results not only identify a reliable marker for defining HECs, but also establish a robust platform for dissecting hematopoiesis in vitro, which might lead to the generation of HSCs in vitro.