The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway, which utilizes β-catenin to relay the signal, have been demonstrated to regulate HSC function. In this study, we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation, such as increased expression of myc . Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G state. From these data, we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway, resulting in a pool of quiescent HSCs.