Certain bone marrow (BM) Focal Adhesion Kinase (FAK) niches are essential for hematopoietic stem cell (HSC) perform in the course of the two regular hematopoiesis and in stem cell transplantation therapy. We show that the guidance molecule Robo4 functions to exclusively anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and significantly lowered long-term reconstitutionetc capability though retaining multilineage possible. Cxcr4, a essential regulator of HSC place, is upregulated in Robo4(-/-) HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of each Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Remarkably, we discovered that WT HSCs express quite reduced ranges of Cxcr4 and reply poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive accessibility to restricted stem cell niches, and we propose Robo4 being a therapeutic target in HSC transplantation treatment.