Blood flow has long been thought to be important for vessel development and function, but its role in hematopoietic stem cell (HSC) development is not yet fully understood. Here, we take advantage of zebrafish embryos with circulation defects that retain relatively normal early development to illustrate the combinatorial roles of genetic and hemodynamic forces in HSC development. We show that blood flow is not required for initiation of HSC gene expression, but instead is indispensable for its maintenance. Knockdown of klf2a mimics the silent heart (sih/tnnt2a) phenotype while overexpression of klf2a in tnnt2a morphant embryos can rescue HSC defects, suggesting that klf2a is a downstream mediator of blood flow. Furthermore, the expression of nitric oxide (NO) synthase (nos) was reduced in klf2a knockdown embryos, and chromatin immunoprecipitation (ChIP) analysis showed that endogenous Klf2a is bound to the promoters of nos genes in vivo, indicating direct gene regulation. Finally, administration of the NO agonist SNAP can restore HSC development in tnnt2a- and klf2a morphants, suggesting that NO signalling is downstream of Klf2a which is induced by hemodynamic forces. Taken together, we have demonstrated that blood flow is essential for HSC development and is mediated by a klf2a-NO signalling cascade in zebrafish.