TGF-β has been shown to play a central role in regulating inflammatory responses; thus, understanding the factors involved in the generation of TGF-β-producing cells could lead to interventions that are useful in effecting disease progression. In initial studies, the capacity of naive CD4+ T cells from TCR transgenic (Tg) mice to produce TGF-β following primary and secondary stimulation was assessed. TGF-β, IL-4, or IFN-γ production could not be detected from highly purified naive CD4+/lymphocyte endothelial cell adhesion molecule (LECAM)-1 high cells following primary stimulation for 36 h with plate-bound anti-CD3, anti-CD28, and IL-2. This population was subsequently used to study the differentiation of TGF-β-producing CD4+ T cells. In further studies, naive CD4+/LECAM-1 high cells from TCR transgenic mice of both the BALB/c and B10. A backgrounds were stimulated with T-depleted spleen cells (TDS) and specific peptide in the presence of various cytokines and/or cytokine antagonists for 5 days, restimulated, and TGF-β, IL-4, and IFN-γ production were measured. Priming conditions favoring high IL-4 production and/or low IFN-γ production greatly enhanced TGF-β production in secondary cultures. Furthermore, the presence of IL-10 in cultures was associated with an increase in TGF-β production following restimulation. The importance of IL-4 and IFN-γ in regulating TGF-β production was confirmed in studies showing that cells from IFN-γ−/− mice produced more TGF-β, while cells from IL-4−/− mice produced less TGF-β compared with wild-type controls. Finally, the addition of exogenous TGF-β to priming cultures significantly enhanced the production of TGF-β upon restimulation, demonstrating that TGF-β has a role in self-regulating its own production.