Proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) within bone marrow (BM) niches are regulated by adhesion molecules and cytokines produced by mesenchymal stem/progenitor cells (MPC) and osteoblasts (OB). HSPCs that egresses to peripheral blood are widely used for transplant and granulocyte-colony stimulating factor (G-CSF) is used clinically to induce mobilization. The mechanisms, through which G-CSF regulates HSPC trafficking, however, are not completely understood. Herein we show that G-CSF-driven neutrophil expansion alters the BM niche that leads to HSPC mobilization. Alcam(-)Sca-1(+)MPC and Alcam(+)Sca-1(-) OB are reduced coincident with mobilization, which correlates inversely with BM neutrophil expansion. In mice made neutropenic by the neutrophil-specific anti-Ly6G antibody, G-CSF-mediated reduction in MPC and OB is attenuated and mobilization reduced without an effect on monocytes/macrophages. Neutrophils, expanded in response to G-CSF-induced MPC and OB apoptosis leading to reduced production of BM HSPC retention factors, including stromal cell-derived factor-1, stem cell factor and vascular cell adhesion molecule-1. Blockade of neutrophil reactive oxygen species attenuates G-CSF-mediated MPC and OB apoptosis. These data show that the expansion of BM neutrophils by G-CSF contributes to the transient degradation of retention mechanisms within the BM niche, facilitating enhanced HSPC egress/mobilization.