Long-term treatment of mice or humans with granulocyte colony-stimulating factor (G-CSF) is associated with a clinically significant osteopenia characterized by increased osteoclast activity and number. In addition, recent reports have observed a decrease in number of mature osteoblasts during G-CSF administration. However, neither the extent of G-CSF's suppressive effect on the osteoblast compartment nor its mechanisms are well understood. Herein, we show that short-term G-CSF treatment in mice leads to decreased numbers of endosteal and trabecular osteoblasts. The effect is specific to mature osteoblasts, because bone-lining cells, osteocytes, and periosteal osteoblasts are unaffected. G-CSF treatment accelerates osteoblast turnover in the bone marrow by inducing osteoblast apoptosis. In addition, whereas G-CSF treatment sharply increases osteoprogenitor number, differentiation of mature osteoblasts is impaired. Bone marrow transplantation studies show that G-CSF acts through a hematopoietic intermediary to suppress osteoblasts. Finally, G-CSF treatment, through suppression of mature osteoblasts, also leads to a marked decrease in osteoprotegerin expression in the bone marrow, whereas expression of RANKL remains relatively constant, suggesting a novel mechanism contributing to the increased osteoclastogenesis seen with long-term G-CSF treatment. In sum, these findings suggest that the hematopoietic system may play a novel role in regulating osteoblast differentiation and apoptosis during G-CSF treatment.