Friend leukemia virus integration 1 (Fli-1) is an ETS transcription factor and a critical regulator of inflammatory mediators, including MCP-1, CCL5, IL-6, G-CSF, CXCL2, and caspase-1. GM-CSF is a regulator of granulocyte and macrophage lineage differentiation and a key player in the pathogenesis of inflammatory/autoimmune diseases. In this study, we demonstrated that Fli-1 regulates the expression of GM-CSF in both T cells and endothelial cells. The expression of GM-CSF was significantly reduced in T cells and endothelial cells when Fli-1 was reduced. We found that Fli-1 binds directly to the GM-CSF promoter using chromatin immunoprecipitation assay. Transient transfection assays indicated that Fli-1 drives transcription from the GM-CSF promoter in a dose-dependent manner, and mutation of the Fli-1 DNA binding domain resulted in a significant loss of transcriptional activation. Mutation of a known phosphorylation site within the Fli-1 protein led to a significant increase in GM-CSF promoter activation. Thus, direct binding to the promoter and phosphorylation are two important mechanisms behind Fli-1–driven activation of the GM-CSF promoter. In addition, Fli-1 regulates GM-CSF expression in an additive manner with another transcription factor Sp1. Finally, we demonstrated that a low dose of a chemotherapeutic drug, camptothecin, inhibited expression of Fli-1 and reduced GM-CSF production in human T cells. These results demonstrate novel mechanisms for regulating the expression of GM-CSF and suggest that Fli-1 is a critical druggable regulator of inflammation and immunity.