The use of nonmyeloablative conditioning prior to bone marrow transplantation is an important component of transplantation-based therapies for nonmalignant blood diseases. In this study, treatment of recipient mice with granulocyte colony-stimulating factor (G-CSF) prior to low-dose total body irradiation (LD-TBI) enhanced long-term engraftment of freshly isolated congenic marrow 1.5- to 2-fold more than treatment with LD-TBI alone. This combined regimen was also evaluated in a mouse model of X-linked chronic granulomatous disease (X-CGD), where neutrophils have a defective NADPH oxidase due to genetic deletion of the gp91(phox) subunit. Long-term engraftment of male X-CGD bone marrow cells cultured ex vivo for retroviral transduction of gp91(phox) was enhanced by approximately 40% when female X-CGD recipients were pretreated with G-CSF prior to 300 cGy. These data confirm that sequential treatment with G-CSF and LD-TBI prior to transplantation increases long-term engraftment of donor marrow, and they extend this approach to transplantation of murine donor marrow cultured ex vivo for gene transfer. Additional studies showed that the administration of G-CSF prior to LD-TBI did not alter early homing of donor marrow cells. However, the combined regimen significantly decreased the content of long-term repopulating cells in recipient marrow compared with LD-TBI alone, as assessed in competitive assays, which may contribute to the enhanced engraftment of donor marrow cells. Disclosure of potential conflicts of interest is found at the end of this article.