The differentiation of T helper (Th) cells is regulated by members of the signal transducer and activator of transcription (STAT) family of signaling molecules. We have generated mice lacking both Stat4 and Stat6 to examine the ability of Th cells to develop in the absence of these two transcription factors. Stat4, Stat6^−/− lymphocytes fail to differentiate into interleukin (IL)-4–secreting Th2 cells. However, in contrast to Stat4^−/− lymphocytes, T cells from Stat4, Stat6^−/− mice produce significant amounts of interferon (IFN)-γ when activated in vitro. Although Stat4, Stat6^−/− lymphocytes produce less IFN-γ than IL-12–stimulated control lymphocytes, equivalent numbers of IFN-γ–secreting cells can be generated from cultures of Stat4, Stat6^−/− lymphocytes activated under neutral conditions and control lymphocytes activated under Th1 cell–promoting conditions. Moreover, Stat4, Stat6^−/− mice are able to mount an in vivo Th1 cell–mediated delayed-type hypersensitivity response. These results support a model of Th cell differentiation in which the generation of Th2 cells requires Stat6, whereas a Stat4-independent pathway exists for the development of Th1 cells.