STAT4 is an essential transcription factor for Th1 cell development. IL-12 and IFN-α both activate STAT4, but with different kinetics. In this study we compared their capacities to drive differentiation of human naive Th cells toward the Th1 phenotype. The Th1-polarizing activity of IFN-α was much weaker than that of IL-12, correlating with a marked difference in the kinetics of STAT4 activation; the response to IL-12 was sustained (>48 h), whereas the response to IFN-α was transient (4 h). The continuous presence of IL-12 was required for sustained STAT4 activation. Similarly, optimal Th1 polarization was only achieved upon prolonged exposure to IL-12 and could not be induced by a transient IL-12 pulse. Furthermore, the cytokine IL-2 potentiated sustained IL-12/STAT4 responses through up-regulation of IL-12R expression and synergized with IL-12 in driving Th1 cell development. Transient IFN-α responses, on the other hand, were not prolonged by IL-2. IFN-α treatment induced down-regulation of IFN-αβ receptor subunit 1, rendering cells refractory to IFN-α, but did not trans-inhibit the IL-12/STAT4 response. These data indicate that sustained IL-12 signaling is essential for optimal Th1 cell development and that transient activation of STAT4 in response to IFN-α may explain the poor Th1-polarizing capacity of this cytokine. Collectively these data show that the duration of cytokine signaling is important for determining the biological response.