The bone marrow microenvironment/niche plays a key role in regulating hematopoietic stem and progenitor cell (HSPC) activities, however mechanisms regulating niche cell function are not well understood. In this study, we show that niche intrinsic expression of the CXCR4 chemokine receptor critically regulates HSPC maintenance during study-state, and promotes early hematopoietic regeneration after myeloablative irradiation. At steady-state, chimeric mice with wild-type (WT) HSPC and marrow stroma that lack CXCR4 show decreased HSPC quiescence, and their repopulation capacity was markedly reduced. Mesenchymal stromal cells (MSC) were significantly reduced in the bone marrow (BM) of CXCR4 deficient mice, which was accompanied by decreased levels of the HSPC supporting factors stromal cell-derived factor-1 (SDF-1) and stem cell factor (SCF). CXCR4 also plays a crucial role in survival and restoration of BM stromal cells after myeloablative irradiation, where loss of BM stromal cells was more severe in CXCR4 deficient mice compared to WT mice. In addition, transplantation of WT donor HSPC into CXCR4 deficient recipient mice demonstrated reduced HSPC homing and early hematopoietic reconstitution. We found that CXCR4 signaling attenuates irradiation-induced BM stromal cell loss by up-regulating the expression of the anti-apoptotic protein Survivin via the PI3K pathway. Our study suggests that SDF-1-CXCR4 signaling in the stromal microenvironment cells plays a crucial role in maintenance of HSPCs during homeostasis, and promotes niche regeneration and early hematopoietic reconstitution after transplantation. Modulation of CXCR4 signaling in the HSPC microenvironment could be a means to enhance hematopoietic recovery after clinical hematopoietic cell transplantation. © AlphaMed Press 2020 SIGNIFICANCE STATEMENT: Our study identify how the bone marrow niche intrinsic CXCR4-SDF-1 axis supports HSPC maintenance and retention under steady-state, and promotes early hematopoietic recovery after myeloablation. Our findings suggest that targeted modulation of CXCR4 signaling in bone marrow niche may provide a novel approach to alleviate the irradiation/chemotherapy-induced bone marrow stroma damage, to enhance blood cell production in patients undergoing stem cell transplantation for several hematological complications including leukemia, lymphoma and aplastic anemia, as well as metabolic disorders such as diabetes.© 2020 AlphaMed Press.