Emerging evidence suggests that adenomatous polyposis coli (Apc) plays a critical role in the maintenance of hematopoietic stem/progenitor cells (HSCs/HPCs). The molecular pathways responsible for the function of Apc in HSCs/HPCs remain unclear. By genetic approach, we demonstrated that inactivation of β-catenin rescued the exhaustion of Apc-deficient HSCs/HPCs, thereby preventing bone marrow failure in Apc-deficient mice. β-catenin loss inhibited the excessive proliferation and apoptosis of Apc-deficient HSCs/HPCs, as well as their defects in myeloid and erythroid differentiation. In addition, loss of β-catenin reversed the down-regulation of Cdkn1a, Cdkn1b, and Mcl1 induced by Apc ablation in Lin⁻Sca⁺c-Kit⁺. In assays of long-term stem cell function, the HSCs with deficiency of both Apc and β-catenin displayed a significantly enhanced self-renewal capacity compared with β-catenin–deficient and control HSCs. Our findings suggest that Apc regulates the survival, proliferation, and differentiation of HSCs/HPCs largely through a β-catenin–mediated pathway. They also indicate that multiple downstream targets of Apc including β-catenin may coordinately regulate HSC self-renewal.