CD4+ T helper cells with the ability to produce effector cytokines provide host protection by regulating immune responses against pathogens. In contrast, these cells are responsible for the development of various inflammatory disease if not regulated properly. Previous studies using naïve CD4+ T cell activation in vitro have shown the requirement of various cytokine combinations in addition to TCR activation to differentiate naïve CD4+ T cells into various effector T helper lineages. The recently discovered CD4+ T helper subset is IL-9-producing Th9 cells. Since Th9 cell differentiation in vitro is essential in understanding the molecular mechanism in regulating Th9 cell development, it is critical to develop a basic protocol in polarizing naïve CD4+ T cells to Th9 cells in vitro. Here we describe a simple method for Th9 cell culture conditions in vitro that can be used for other molecular analyses.