We report here that Janus kinase 3 (Jak3) is a primary response gene for interleukin-6 (IL-6) in macrophage differentiation, and ectopic overexpression of Jak3 accelerates monocytic differentiation of normal mouse bone marrow cells stimulated with cytokines. Furthermore, we show that incubation of normal mouse bone marrow cells with a JAK3-specific inhibitor results in profound inhibition of myeloid colony formation in response to granulocyte-macrophage colony-stimulating factor or the combination of stem cell factor, IL-3, and IL-6. In addition, mutagenesis of the Jak3 promoter has revealed that Sp1 binding sites within a -67 to -85 element and a signal transducer and activator of transcription (Stat) binding site at position -44 to -53 are critical for activation of Jak3 transcription in murine M1 myeloid leukemia cells stimulated with IL-6. Electrophoretic mobility shift assay (EMSA) analysis has demonstrated that Sp1 can bind to the -67 to -85 element and Stat3 can bind to the -44 to -53 STAT site in IL-6-stimulated M1 cells. Additionally, ectopic overexpression of Stat3 enhanced Jak3 promoter activity in M1 cells. This mechanism of activation of the murine Jak3 promoter in myeloid cells is distinct from a recently reported mechanism of activation of the human JAK3 promoter in activated T cells.