Wnt/Fzd signaling is necessary throughout development and homeostasis. At least two different Wnt signals have been shown to be necessary for the formation of hematopoietic stem cells (HSCs). In particular, Wnt16 signaling is required for the specification of HSCs, and Wnt9a is required for the amplification of HSCs, in two distinct developmental events. Using the zebrafish, paired with cell culture models, we previously identified that Wnt9a and its cognate Frizzled (Fzd) receptor, Fzd9b, are required for HSC proliferation. We are now focused on identifying other cognate Wnt/Fzd pairings required for HSC ontogeny. We have identified the Fzd5 receptor as necessary for specification of HSCs. Loss of this receptor via morpholino knockdown leads to fewer HSCs in the floor of the dorsal aorta at 28 hours post fertilization. Furthermore, rag1+ thymic cells, which are descendants of HSCs, are ablated in these morphants, indicative of a failure of HSC development. These findings were specific to HSCs; we observed normal development of primitive blood and other structures associated with HSC development. To validate these findings further, we have begun establishing germline fzd5 zebrafish mutants with CRISPR/Cas9. In addition, to explore the relationship between Wnt16 and Fzd5 we tested complementation of the two genes by utilizing suboptimal dosages of morpholinos targeting wnt16 and fzd5 and observed HSCs were again reduced when compared to single morpholino suboptimal dose controls.