During development, haemogenesis occurs invariably at sites of vasculogenesis. 
Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal 
part of the dorsal aorta generate haematopoietic stem cells and are referred to 
as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted 
to this domain, and how the morphological transformation from endothelial to 
haematopoietic is controlled are unknown. We show here that HoxA3, a gene 
uniquely expressed in the embryonic but not yolk sac vasculature, restrains 
haematopoietic differentiation of the earliest endothelial progenitors, and 
induces reversion of the earliest haematopoietic progenitors into CD41-negative 
endothelial cells. This reversible modulation of endothelial-haematopoietic 
state is accomplished by targeting key haematopoietic transcription factors for 
downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through 
loss-of-function, and gain-of-function epistasis experiments, and the 
identification of antipodally regulated targets, we show that among these 
factors, Runx1 is uniquely able to erase the endothelial program set up by 
HoxA3. These results suggest both why a frank endothelium does not precede 
haematopoiesis in the yolk sac, and why haematopoietic stem cell generation 
requires Runx1 expression only in endothelial cells.