Definitive hematopoietic stem/progenitor cells (HSPCs) are produced from a specialized population of endothelial cells in the embryonic aorta called hemogenic endothelial cells. This process, known as the endothelial-to-hematopoietic transition (EHT), is conserved across vertebrates, and is followed by HSPC differentiation in all blood lineages. Single-cell methods have uncovered substantial heterogeneity in lineage priming within nascent HSPCs from the aortic endothelium, however it is currently unknown how this diverse differentiation capacity is conferred. Such understanding could have important ramifications for bone marrow transplantation where heterogeneity in HSPC behavior is observed. Here we found that microRNA (miR) loss of function zebrafish mutant, miR-128 (miR-128-/-), has increased EHT, resulting in supernumerary lymphoid and erythroid primed HSPCs and relative mature cells. Correlatively, miR-128-deficient endothelium derived from human pluripotent stem cells show similar defective EHT, supporting miR-128’s functional conservation in human. Transcriptomic analysis of endothelial cells from wild type and miR-128-/- embryos revealed putative miR-128 target genes, cskn1a and jag1b, involved in the inhibition of the two master EHT signaling pathways, Wnt and Notch respectively. Additionally, responsive transgenic lines and single cell RNA sequencing revealed that Wnt and Notch signaling activity are decreased specifically in pre-hemogenic endothelium of miR-128-/-. Chemical inhibition of Wnt or de-repression of cskn1a promoted HSPCs with erythroid lineage commitment, while lymphoid cells number increased after de-repression of jag1b or blockage of Notch activity at the onset of EHT. This study sheds light on how the HSPC heterogeneity in lineage priming is programmed prior to endothelial transdifferentiation by miR-128-co regulation of Wnt and Notch. Definitive hematopoietic stem/progenitor cells (HSPCs) are produced from a specialized population of endothelial cells in the embryonic aorta called hemogenic endothelial cells. This process, known as the endothelial-to-hematopoietic transition (EHT), is conserved across vertebrates, and is followed by HSPC differentiation in all blood lineages. Single-cell methods have uncovered substantial heterogeneity in lineage priming within nascent HSPCs from the aortic endothelium, however it is currently unknown how this diverse differentiation capacity is conferred. Such understanding could have important ramifications for bone marrow transplantation where heterogeneity in HSPC behavior is observed. Here we found that microRNA (miR) loss of function zebrafish mutant, miR-128 (miR-128-/-), has increased EHT, resulting in supernumerary lymphoid and erythroid primed HSPCs and relative mature cells. Correlatively, miR-128-deficient endothelium derived from human pluripotent stem cells show similar defective EHT, supporting miR-128’s functional conservation in human. Transcriptomic analysis of endothelial cells from wild type and miR-128-/- embryos revealed putative miR-128 target genes, cskn1a and jag1b, involved in the inhibition of the two master EHT signaling pathways, Wnt and Notch respectively. Additionally, responsive transgenic lines and single cell RNA sequencing revealed that Wnt and Notch signaling activity are decreased specifically in pre-hemogenic endothelium of miR-128-/-. Chemical inhibition of Wnt or de-repression of cskn1a promoted HSPCs with erythroid lineage commitment, while lymphoid cells number increased after de-repression of jag1b or blockage of Notch activity at the onset of EHT. This study sheds light on how the HSPC heterogeneity in lineage priming is programmed prior to endothelial transdifferentiation by miR-128-co regulation of Wnt and Notch.