T-bet is an important Th1 driving transcription factor regulated by IFN-gamma/STAT1 pathway. T-bet turns on IFN-gamma transcription in CD4+ T cells and T-bet-deficient cells fail to differentiate to Th1 direction. Previous reports have characterized function of T-bet mainly in murine cells and very little is known about its functions in human cells. Here, we studied T-bet expression kinetics in parallel with GATA3 during Th1/Th2 polarization. We demonstrate that in addition to CD3/CD28 activation, cytokines IL-12 and IFN-alpha in the presence of neutralizing anti-IFN-gamma enhanced T-bet mRNA and protein expression in human CD4+ cells. T-bet is known to be a potent inducer of IFN-gamma. Even though IFN-gamma and IL-12 stimulation induced similar levels of T-bet protein in human CD4+ cells, IFN-gamma-treated cells produced considerably less IFN-gamma than cells treated with IL-12. Therefore, high T-bet protein expression does not necessarily correlate with IFN-gamma production. In addition, we show that the immunosuppressive cytokine TGF-beta inhibits T-bet and GATA3 protein expression only if it is present prior to primary T cell activation and is maintained in the cultures during the early polarization of Th1/Th2 cells. In conclusion, we report new insights into the cytokine regulation of T-bet in human CD4+ T cells.