T cell activation required for host defense against infection is an intricately regulated and precisely controlled process. Although in vitro studies indicate that three distinct stimulatory signals are required forT cell activation, the precise contribution of each signal in regulating T cell proliferation and differentiation after in vivo infection is unknown. In this study, altered peptide ligands (APLs) derived from the protective Salmonella-specific FliC Ag and CD4+ T cells specific for the immune-dominant FliC431-439 peptide within this Ag were used to determine how changes in TCR stimulation impact CD4+ T cell proliferation, differentiation, and protective potency. To explore the prevalence and potential use of altered TCR stimulation by bacterial pathogens, naturally occurring APLs containing single amino acid substitutions in putative TCR contact residues within the FliC431-439 peptide were identified and used for stimulation under both noninfection and infection conditions. On the basis of this analysis, naturally-occurring APLs that prime proliferation of FliC-specific CD4+ T cells either more potently or less potently compared with the wild-type FliC431-439 peptide were identified. Remarkably, despite these differences in proliferation, all of the APLs primed reduced IFN-γ production by FliC431-439-specific CD4 + T cells after stimulation in vivo. Moreover, after expression of the parental FliC431-439 peptide or each APL in recombinant Listeria monocytogenes, only CD4+ T cells stimulated with the wild-type FliC431-439 peptide conferred significant protection against challenge with virulent Salmonella. These results reveal important and unanticipated roles for TCR stimulation in controlling pathogen-specific CD4+ T cell proliferation, differentiation, and protective potency.