Granulocyte colony-stimulating factor (G-CSF) is a pleiotropic cytokine that drives the production, survival, differentiation, and inflammatory functions of granulocytes. Reactive oxygen species (ROS) provide a major thrust of the inflammatory response, though excessive ROS production may be deleterious. G-CSF stimulation showed a time and dose-dependent increase in ROS production, correlating with activation of the Src kinase Lyn and Akt. Inhibition of Lyn, PI 3-kinase, and Akt, but not Erk1/2, abrogated G-CSF-induced ROS production. Following G-CSF stimulation, neutrophils from bone marrow of lyn-/- mice produced less ROS than wild-type littermates. G-CSF induced both serine phosphorylation and membrane translocation of p47(phox), a subunit of NADPH oxidase. The NADPH oxidase inhibitor DPI blocked the G-CSF-induced ROS production in G-CSFR expressing Ba/F3 cells. Because patients with a truncated G-CSF Receptor have a high risk of developing acute myeloid leukemia (AML), we hypothesized that dysregulation of ROS production might contribute to leukemogenesis. Cells expressing the truncated G-CSF Receptor produced more ROS than those with the full-length receptor. G-CSF-induced ROS production was also enhanced in bone marrow-derived neutrophils expressing G-CSFRDelta715, a truncated receptor. The antioxidant N-Acetyl-L-Cysteine diminished G-CSF-induced ROS production and cell proliferation by inhibiting Akt activation. These data suggest that the Lyn-PI3K-Akt pathway plays a critical role in the G-CSF-induced production of ROS. Hyperactivation of Lyn with subsequent augmented ROS production may constitute one mechanism by which AML arises. These studies suggest that one beneficial effect of therapeutic targeting of Lyn-PI 3K-kinase-Akt cascade is blocking ROS production.