Naive CD4+ T cells differentiate into various effector Th subsets depending on the Ags and cytokine microenvironment they encounter. IL-9-secreting Th9 cells are the most recent Th subset to be described. PU.1, one of the transcription factors required for the development of Th9 cells, binds to the Il9 gene. In this study, we show that PU.1 increases histone acetylation at the Il9 locus through direct interactions with histone acetyltransferases. In the absence of PU.1, there is decreased association of Gcn5 and p300/CBP associated factor and increased association of histone deacetylases at the Il9 locus in Th9 cells. Inhibition of histone deacetylase activity augments PU.1-dependent IL-9 production. PU.1 forms a complex with Gcn5, and inhibition of the expression of Gcn5 results in reduced IL-9 production. Moreover, the effects of Gcn5 on IL-9 production are specific as the production of IL-10 and IL-21, two additional cytokines produced by Th9 cells, is not altered after decreased Gcn5 expression. Together, these data define a PU.1-dependent mechanism for altered histone acetylation and expression of the Il9 locus in Th9 cells.