Ionizing irradiation (IR) causes bone marrow (BM) injury, with senescence and impaired self-renewal of hematopoietic stem cells (HSCs), and inhibiting Wnt signaling could enhance hematopoietic regeneration and survival against IR stress. However, the underlying mechanisms by which a Wnt signaling blockade modulates IR-mediated damage of BM HSCs and mesenchymal stem cells (MSCs) are not yet completely understood. We investigated the effects of osteoblastic Wntless (Wls) depletion on total body irradiation (TBI, 5 Gy)-induced impairments in hematopoietic development, MSC function, and the BM microenvironment using conditionalWlsknockout mutant mice (Col-Cre;Wlsfl/fl) and their littermate controls (Wlsfl/fl). OsteoblasticWlsablation itself did not dysregulate BM frequency or hematopoietic development at a young age. Exposure to TBI at 4 weeks of age induced severe oxidative stress and senescence in the BM HSCs ofWlsfl/flmice but not in those of theCol-Cre;Wlsfl/flmice. TBI-exposedWlsfl/flmice exhibited greater impairments in hematopoietic development, colony formation, and long-term repopulation than TBI-exposedCol-Cre;Wlsfl/flmice. Transplantation with BM HSCs or whole BM cells derived from the mutant, but notWlsfl/flmice, protected against HSC senescence and hematopoietic skewing toward myeloid cells and enhanced survival in recipients of lethal TBI (10 Gy). Unlike theWlsfl/flmice, theCol-Cre;Wlsfl/flmice also showed radioprotection against TBI-mediated MSC senescence, bone mass loss, and delayed body growth. Our results indicate that osteoblasticWlsablation renders BM-conserved stem cells resistant to TBI-mediated oxidative injuries. Overall, our findings show that inhibiting osteoblastic Wnt signaling promotes hematopoietic radioprotection and regeneration.