Natural killer T (NKT) cells are a highly conserved subset of T cells that have been shown to play a critical role in suppressing T helper cell type 1–mediated autoimmune diseases and graft versus host disease in an interleukin (IL)-4–dependent manner. Thus, it is important to understand how the development of IL-4– versus interferon (IFN)-γ–producing NKT cells is regulated. Here, we show that NKT cells from adult blood and those from cord blood undergo massive expansion in cell numbers (500–70,000-fold) during a 4-wk culture with IL-2, IL-7, phytohemagglutinin, anti-CD3, and anti-CD28 mAbs. Unlike adult NKT cells that preferentially produce both IL-4 and IFN-γ, neonatal NKT cells preferentially produce IL-4 after polyclonal activation. Addition of type 2 dendritic cells (DC2) enhances the development of neonatal NKT cells into IL-4⁺IFN-γ⁻ NKT2 cells, whereas addition of type 1 dendritic cells (DC1) induces polarization towards IL-4⁻IFN-γ⁺ NKT1 cells. Adult NKT cells display limited plasticity for polarization induced by DC1 or DC2. Thus, newly generated NKT cells may possess the potent ability to develop into IL-4⁺IFN-γ⁻ NKT2 cells in response to appropriate stimuli and may thereafter acquire the tendency to produce both IL-4 and IFN-γ.