Abstract Interleukin-9 (IL-9) is a multifunctional cytokine involved in proliferation, differentiation, and effector functions of various cell types that include T cells, B cells, mast cells, eosinophiles neutrophils, and epithelial cells. In previous studies, Th2 cells were shown to produce IL-9. Recent studies suggest that IL-9 can also be produced by so-called Th9 cells. In this study, we report that TGF-beta is an essential factor for in vitro cultured human peripheral blood mononuclear cells (PBMC) into subsets that secrete IL-9. PBMC were activated in the presence of anti-CD3/CD28 antibodies and polarizing cytokines with or without the addition of TGF-beta. Using flow cytometry to detect both intracellular IL-9 expression and soluble IL-9 secretion, we found that IL-9 was only produced in the presence of TGF-beta. Addition of other cytokines, especially IL-4 further increased IL-9 production. Interestingly, a subset of CD4+IL-9+ T cells was seen to also express IL-17, but not IL-4, IL-10 or IL-12. Additional studies will address the plasticity between the Th9 and Th17 CD4 T cell subsets.