Hematopoietic and vascular developments are closely related physiological processes during vertebrate embryogenesis.Recently, endothelial to hematopoietic transition (EHT) is demonstrated to be essential for hematopoietic cells induction, but its underlying regulatory mechanisms still remain poorly understood.Here we show that thrombin receptor (F2r), a protease activated G protein-coupled receptor required for vascular development, negatively regulates hematopoietic differentiation of mouse embryonic stem cells.Mechanistic studies reveal that RhoA/ROCK pathway mediates F2r signaling and inhibits EHT in vitro.Furthermore, F2r-RhoA/ROCK pathway negatively regulates zebrafish definitive hematopoiesis, EHT and hematopoietic stem cells induction in vivo.Taken together, this study demonstrates a fundamental role of F2r-RhoA/ROCK pathway in vertebrate hematopoiesis and EHT, as well as an important molecular mechanism keeping the balance between hematopoietic and vascular development.