Detailed understanding of the genetic regulation of haematopoiesis is essential for blood cell therapies and to faithfully model haematologic disease. Unlike in the mouse, this knowledge is lacking in humans. To address this, we followed endothelial (SOX17) and haematopoietic (RUNX1C) development using in vitro differentiation of human pluripotent stem cells (hPSCs). Mimicking yolk sac haematopoiesis, we identified SOX17-CD34+CD43- endothelial-like cells as the major source of haematopoietic progeny while SOX17+CD34+CD43- cells mostly formed endothelium. Deletion of RUNX1 permitted a single wave of GFI1/1B-dependent yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Deletion of SOX17 or all Group F SOX genes (SOX7, SOX17, SOX18) did not influence yolk sac-like haematopoiesis, but severely perturbed the development of AGM-like vessels and reduced their haemogenic capacity. This phenotype was largely mediated by reduced NOTCH signaling and recapitulated by inhibiting γ-secretase. Our data extend mouse studies, identifying unique and distinct requirements for RUNX1 and SOX genes during human haematopoiesis.