Haematopoiesis in adult animals is maintained by haematopoietic stem cells (HSCs), which self-renew and can give rise to all blood cell lineages. The AGM region is an important intra-embryonic site of HSC development and a wealth of evidence indicates that HSCs emerge from the endothelium of the embryonic dorsal aorta and extra-embryonic large arteries. This, however, is a stepwise process that occurs through sequential upregulation of CD41 and CD45 followed by emergence of fully functional definitive HSCs. Although largely dispensable at later stages, the Runx1 transcription factor is crucially important during developmental maturation of HSCs; however, exact points of crucial involvement of Runx1 in this multi-step developmental maturation process remain unclear. Here, we have investigated requirements for Runx1 using a conditional reversible knockout strategy. We report that Runx1 deficiency does not preclude formation of VE-cad+CD45−CD41+ cells, which are phenotypically equivalent to precursors of definitive HSCs (pre-HSC Type I) but blocks transition to the subsequent CD45+ stage (pre-HSC Type II). These data emphasise that developmental progression of HSCs during a very short period of time is regulated by precise stage-specific molecular mechanisms.