CD4 + Th cells can have cytotoxic activity against cells displaying relevant peptide-MHC class II (p:MHCII) ligands. Cytotoxicity may be a property of Th1 cells and depends on perforin and the Eomes transcription factor. We assessed these assertions for polyclonal p:MHCII-specific CD4+ T cells activated in vivo in different contexts. Mice immunized with an immunogenic peptide in adjuvant or infected with lymphocytic choriomeningitis virus or Listeria monocytogenes bacteria induced cytotoxic Th cells that killed B cells displaying relevant p:MHCII complexes. Cytotoxicity was dependent on Fas expression by target cells but was independent of Eomes or perforin expression by T cells. Although the priming regimens induced different proportions of Th1, Th17, regulatory T cells, and T follicular helper cells, the T cells expressed Fas ligand in all cases. Reciprocally, Fas was upregulated on target cells in a p:MHCII-specific manner. These results indicate that many Th subsets have cytotoxic potential that is enhanced by cognate induction of Fas on target cells.