Recent progress by versatile approaches supports the new hypothesis that multi-potent hematopoietic stem cells(HSCs) are directly formed from a rare population of endothelial cells in mid-gestation mouse embryos.This process is therefore known as the endothelial-tohematopoietic transition(EHT).Nevertheless,there is no functional evidence that documents the HSC transition from purified endothelial cells.In this study,we developed an OP9-DL1-based co-culture system that was able to facilitate the HSC specification and/or expansion in vitro of mouse embryonic day 10.5(E10.5) Tie2~+ cells remarkably.Then,the immunophenotypically defined endothelial cells were harvested by a combination of surface markers(Flk1~+CD31~+CD41~-CD45~-Ter119~-) from the caudal half of E10.0-E11.0 mouse cmbryos.The transplantation of the endothelia/OP9-DL1 co-cultures led to long-term,high-level,multi-lineage,and multi-organ hematopoietic reconstitution in the irradiated adult recipients.The induced HSC activity was initially observed at E10.5,and a significant increase was detected at E11.0,which suggests a temporally specific regulation.Taken together,for the first time,we provide functional evidence showing the HSC potential of purified embryonic endothelial cells,which is indispensable for the emerging EHT concept. Moreover,the newly defined co-culture system will aid the exploration of the key molecules governing the HSC transition from embryonic and even postnatal endothelial cells,which has enormous significance in basic and translational research. Recent progress by versatile approaches supports the new hypothesis that multi-potent hematopoietic stem cells (HSCs) are directly formed from a rare population of endothelial cells in mid-gestation mouse embryos. This process is therefore known as the endothelial-to-hematopoietic transition (EHT) There is no functional evidence that documents the HSC transition from purified endothelial cells. In this study, we developed an OP9-DL1-based co-culture system that was able to facilitate the HSC specification and / or expansion in vitro of mouse embryonic day 10.5 (E10.5) Tie2 ~ + cells remarkably. Then, the immunophenotypically defined endothelial cells were harvested by a combination of surface markers (Flk1 ~ + CD31 ~ + CD41 ~ -CD45 ~ -Ter119 ~ -) from the caudal half of E10.0-E11.0 mouse cmbryos. the transplantation of the endothelia / OP9-DL1 co-cultures led to long-term, high-level, multi-lineage, and multi-organ hematopoietic reconstitution in the irradiated adult recipients. induced HSC activity w as initially observed at E10.5, and a significant increase was detected at E11.0, which suggests a temporally specific regulation.Taken together, for the first time, we provide functional evidence showing the HSC potential of purified embryonic endothelial cells, which is indispensable for the emerging EHT concept. Moreover, the newly defined co-culture system will aid the exploration of the key molecules governing the HSC transition from embryonic and even postnatal endothelial cells, which has substantial significance in basic and translational research.