Each of the three Th2 cytokine genes, IL-4, IL-5, and IL-13, has different functions. We hypothesized that Th2 heterogeneity could yield Th2 subpopulations with different cytokine expression and effector functions. Using multiple approaches, we demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5+(IL-5+, IL-4+, IL-13+) and majority IL-5− Th2 (IL-5−, IL-4+, IL-13+) population. IL-5+ Th2 cells comprised only 20% of all Th2 cells. Serial rounds of in vitro differentiation initially yielded IL-5− Th2, but required multiple rounds of differentiation to generate IL-5+ Th2 cells. IL-5+ Th2 cells expressed less CD27 and greater programmed cell death-1 than IL-5− Th2 cells, consistent with their being more highly differentiated, Ag-exposed memory cells. IL-5+ Th2 cells expressed greater IL-4, IL-13, and GATA-3 relative to IL-5− Th2 cells. GATA-3 and H3K4me 3 binding to the IL5 promoter (IL5p) was greater in IL-5+ relative to IL-5− Th2 cells, whereas there was no difference in their binding to the IL4p and IL13p. Conversely, H3K27me 3 binding to the IL5p was greater in IL-5− Th2 cells. These findings demonstrate Th2 lineage heterogeneity, in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. IL-5+ Th2 cells are phenotypically distinct and have epigenetic changes consistent with greater IL5p accessibility. Recurrent antigenic exposure preferentially drives the differentiation of IL-5+ Th2 cells. These results demonstrate that IL-5+ and IL-5− Th2 cells, respectively, represent more and less highly differentiated Th2 cell subpopulations. Such Th2 subpopulations may differentially contribute to Th2-driven pathology.