Interleukin (IL)-9-producing CD4(+) T cells are a novel subset of T helper (Th) cells that develops independently of the Th1, Th2, Th17 and regulatory T-cell lineages. Similar to the murine model, transforming growth factor (TGF)-beta and IL-4 directed human naive CD4(+) T cells to produce IL-9. Whereas IL-4 suppressed TGF-beta-induced Foxp3 expression, TGF-beta failed to inhibit IL-4-mediated upregulation of the Th2 transcription factor GATA-3. Addition of IL-1 beta, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta or IL-21 to Th9-polarizing conditions augmented Th9 differentiation, while the Th1-associated cytokines IFN-gamma and IL-27 partially suppressed IL-9 production. Given that T cells are a primary source of IL-21, IL-21 expression was analyzed under Th9-polarizing conditions in the context of inflammatory cytokines. Surprisingly, type I IFNs induced elevated levels of IL-21, and blockade of IL-21 abrogated their ability to enhance Th9 differentiation. Taken together, these data indicate a complex cytokine network in the regulation of human IL-9-producing CD4(+) T cells.