STAT6 plays critical roles in Th2 cell differentiation, whereas STAT4 and T-bet are important for Th1 cell differentiation. However, it is still largely unknown about the cross talk of these transcription factors during Th1/Th2 cell differentiation. To further address the regulatory mechanisms underlying Th1/Th2 cell differentiation, we generated the mice lacking both STAT6 and T-bet (STAT6(-/-)T-bet(-/-) mice). Importantly, although Th2 cell differentiation was severely and similarly decreased in STAT6(-/-)T-bet(-/-) mice and STAT6(-/-) mice, Th1 cell differentiation was rescued in part in STAT6(-/-)T-bet(-/-) mice as compared with that in T-bet(-/-) mice. While no significant difference was observed in the expression of IL-12Rbeta2 and STAT4 between STAT6(-/-)T-bet(-/-) CD4(+) T cells and T-bet(-/-) CD4(+) T cells, IL-12-induced STAT4 phosphorylation was increased in STAT6(-/-)T-bet(-/-) CD4(+) T cells as compared with that in T-bet(-/-) CD4(+) T cells. These results indicate that STAT6 inhibits T-bet-independent Th1 cell differentiation by suppressing IL-12-STAT4 signaling.