Hmgb3 is an X-linked member of a family of sequence-independent chromatin-binding proteins that is preferentially expressed in hematopoietic stem cells (HSC).Hmgb3-deficient mice (Hmgb3−/Y) contain normal numbers of HSCs, capable of self-renewal and hematopoietic repopulation, but fewer common lymphoid (CLP) and common myeloid progenitors (CMP). In this study, we tested the hypothesis thatHmgb3−/YHSCs are biased toward self-renewal at the expense of progenitor production. Wild-type andHmgb3−/YCLPs and CMPs proliferate and differentiate equallyin vitro, indicating that CLP and CMP function normally inHmgb3−/Ymice.Hmgb3−/YHSCs exhibit constitutive activation of the canonical Wnt signaling pathway, which regulates stem cell self-renewal. Increased Wnt signaling inHmgb3−/YHSCs corresponds to increased expression ofDvl1, a positive regulator of the canonical Wnt pathway. To induce hematopoietic stress and a subsequent response from HSCs, we treatedHmgb3−/Ymice with 5-fluorouracil.Hmgb3−/Ymice exhibit a faster recovery of functional HSCs after administration of 5-fluorouracil compared with wild-type mice, which may be due to the increased Wnt signaling. Furthermore, the recovery of HSC number inHmgb3−/Ymice occurs more rapidly than CLP and CMP recovery. From these data, we propose a model in whichHmgb3is required for the proper balance between HSC self-renewal and differentiation.