We have developed an in vitro differentiation assay in which human naive CD4+ cells are driven toward either the Th1 or Th2 phenotype. We have examined the interrelationships among the expression of IL-2, IL-4, IL-5, IL-10, IL-13, GM-CSF, and IFN-γ in individual cells using intracellular cytokine staining at various times during the differentiation process. We provide direct evidence that the Th2 cytokines IL-4, IL-5, and IL-13, unlike the other cytokines, are regulated by a coordinated mechanism. We also show that IL-10 is expressed by a different subset of cells that is prevalent at early stages of Th2 differentiation, but then diminishes. Additionally we demonstrate that while naive cells can express IL-2 upon activation, they cannot express GM-CSF. Commitment to GM-CSF expression occurs during differentiation in a Th1/Th2 subset-independent manner. Furthermore, we have examined the levels of GATA3, c-Maf, T-bet, and Ets-related molecule during human Th1/Th2 differentiation and suggest that differences in the levels of these critical transcription factors are responsible for commitment toward the Th1 or Th2 lineage.