Model summarizing the dynamic P1-Runx1 and P2-Runx1 expression and requirement during definitive hematopoietic cell generation in the YS and PAS/AGM. See “Discussion” for explanation. In brief, our results suggest at least 1 discrete developmental stage for which Runx1 is critically required: the predominantly P2-Runx1–dependent stage at the onset of Runx1 expression in VE-cadherin⁺ cells. Runx1 is no longer critically required in FL hematopoietic cells (recently reported by Chen et al⁸ ). Yellow indicates endothelial cells; orange, Runx1-expressing hemogenic endothelium (CD41^−/lo; Figure 1A)/early committed hematopoietic cells (CD41⁺); red, Runx1-expressing CD45⁺ hematopoietic cells.