The glioma associated oncogene-1 (GLI1), a downstream effector of the embryonic Hedgehog pathway, was detected in chronic lymphocytic leukemia (CLL), but not normal adult cells. GLI1 activating mutations were identified in 10% of patients with CLL. However, what induces GLI1 expression in GLI1-unmutated CLL cells is unknown. Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in CLL cells and sequence analysis detected putative STAT3-binding sites in the GLI1 gene promoter, we hypothesized that STAT3 induces the expression of GLI1. Western immunoblotting detected GLI1 in CLL cells from 7 of 7 patients, flow cytometry analysis confirmed that CD19+/CD5+ CLL cells co-express GLI1 and confocal microscopy showed co-localization of GLI1 and phosphorylated STAT3. Chromatin immunoprecipitation showed that STAT3 protein co-immunoprecipitated GLI1 as well as other STAT3-regulated genes. Transfection of CLL cells with STAT3-shRNA induced a mark decrease in GLI1 levels, suggesting that STAT3 binds to and induces the expression of GLI1 in CLL cells. An electromobility shift assay confirmed that STAT3 binds, and a luciferase assay showed that STAT3 activates the GLI1 gene. Transfection with GLI1-siRNA significantly increased the spontaneous apoptosis rate of CLL cells, suggesting that GLI1 inhibitors might provide therapeutic benefit to patients with CLL.