Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilization of hematopoietic stem cells (HSC) for autologous transplantation in cancer patients. Increasing the number of available HSC prior to mobilization is a potential strategy to overcome this deficiency. Bone marrow (BM) macrophages regulate HSC niches and promote engraftment of donor HSC post-transplant. In this study, we examined if manipulating macrophages using a modified long-acting Fc fusion protein of CSF1 could influence HSC niches to expand the HSC pool. Acute CSF1-Fc treatment (4 x daily injection) in C57Bl/6 mice induced a robust systemic myeloproliferative response that peaked 1 week post the initial injection. BM and spleen homeostasis were disrupted during peak myelopoiesis, including supressed lymphopoiesis and temporary engraftment of BM hematopoietic stem and progenitor cell (HSPC) in liver. CSF1-Fc effects were transient as by 2 weeks post first CSF1-Fc injection, overall haematopoiesis had normalized, but the number of HSC in BM (1.6 fold), and particularly in spleen (5.9 fold), were significantly elevated compared to controls. This was not due to direct CSF1-Fc actions on HSC as multiple experimental approaches confirmed that HSC and multipotent progenitors do not express CSF1-receptor. The increase in splenic HSC corresponded with a sustained phenotype shift in red pulp macrophages characterized by expression of the BM HSC niche macrophage marker CD169. We observed minimal evidence of in situ macrophage proliferation, indicating specialised adaptation of monocyte-derived macrophages to the CSF1-Fc altered splenic environment. Importantly, CSF1-Fc pre-treatment enhanced HSC mobilization (2.5 fold) into blood after G-CSF treatment when compared to controls. Competitive transplantation demonstrated the CSF1-Fc + G-CSF mobilized blood graft had improved reconstitution potential with no leukocyte lineage bias. Overall, the data suggest that CSF1-Fc treatment resulted in sustained adaptation of resident macrophages with improved capability to support extramedullary HSC niches resulting in a systemic increase in the absolute number of HSC. Therefore, conditioning patients with CSF1 could be a potential therapeutic strategy to overcome poor mobilization and subsequently improve autologous transplantation outcomes.