Hematopoietic cells differentiate during embryogenesis from a population of endothelial cells called hemogenic endothelium (HE) in a process called the endothelial to hematopoietic transition (EHT). The transcription factor Runx1 is required for EHT, but for how long, and which endothelial cells are competent to respond to Runx1 is not known. Here we show that the ability of Runx1 to induce EHT in non-hemogenic endothelial cells depends on the anatomic location of the cell and the developmental age of the conceptus. Ectopic expression of Runx1 in non-hemogenic endothelial cells between embryonic day (E) 7.5 - 8.5 promoted the formation of erythro-myeloid progenitors (EMPs) specifically in the yolk sac, the dorsal aorta, and the heart. The increase in EMPs was accompanied by a higher frequency of HE cells able to differentiate into EMPs in vitro Expression of Runx1 just one day later (E8.5 - E9.5) failed to induce the ectopic formation of EMPs. Therefore, endothelial cells, located in specific sites in the conceptus have a short developmental window of competency during which they can respond to Runx1 and differentiate into blood cells.