Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44^hi memory phenotype CD4⁺ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44^hi CD4⁺ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4⁺ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4⁺ T cells and that CNS-2 active CD44^hi memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4⁺ T cells in allergic responses.