During development, hematopoietic stem cells (HSCs) derive from specialized endothelial cells (ECs), called hemogenic endothelium (HE), via a process called endothelial-to-hematopoietic transition (EHT). While hypoxia inducible factor-1α (HIF-1α) has been reported to positively modulate EHT in vivo, current data indicate the existence of other regulators of this process. Here we show that in zebrafish Hif-2α also positively modulates HSC formation. Specifically, HSC marker gene expression is strongly decreased in hif-1aa;hif-1ab (hif-1α) and in hif-2aa;hif-2ab (hif-2α) zebrafish mutants and morphants. Moreover, live imaging studies reveal a positive role for hif-1α and hif-2α in regulating HE specification. Knockdown of hif-2α in hif-1α mutants leads to a greater decrease in HSC formation, indicating that hif-1α and hif-2α have partially overlapping roles in EHT. Furthermore, hypoxic conditions, which strongly stimulate HSC formation in wild-type animals, have little effect in the combined absence of Hif-1α and Hif-2α function. In addition, we present evidence for Hif and Notch working in the same pathway upstream of EHT. notch1a and notch1b mutants display impaired EHT, which can not be rescued by hypoxia. However, overexpression of the Notch intracellular domain in ECs is sufficient to rescue the hif-1α and hif-2α morphant EHT phenotype, suggesting that Notch signaling functions downstream of the Hif pathway during HSC formation. Altogether, our data provide genetic evidence that Hif-1α and Hif-2α both regulate EHT upstream of Notch signaling.