Abstract A detailed understanding of the molecular and cellular mechanisms that underlie epitope preferences in T cell priming is important for vaccines designed to elicit a broad T cell response. Protein vaccinations generally elicit CD4 T cell responses that are skewed toward a small fraction of epitopes, a phenomenon known as immunodominance. This characteristic of T cell responses, which limits the diversity of CD4 T cell recognition, is generally attributed to intracellular Ag processing. However, we recently discovered that immunodominance hierarchies persist even after vaccination with synthetic peptides. In this study, we probed the regulatory mechanisms that cause diminished CD4 T cell responses to subdominant peptides after such multipeptide immunization in mice. We have found that the delivery of subdominant and dominant epitopes on separate dendritic cells rescues expansion of less favored CD4 T cells. Furthermore, through the use of genetic models and inhibitors, we have found that selective losses in CD4 T cell responses are mediated by an IFN-γ–induced pathway, involving IDO, and that regulatory T cell activities may also regulate preferences in CD4 T cell specificity. We propose that after multipeptide immunization, the expansion and differentiation of dominant T cells initiate complex regulatory events that determine the final peptide specificity of the elicited CD4 T cell response.