We investigated the in vivo priming of IL-17(+) autoreactive T cells in experimental autoimmune uveitis-prone C57BL/6 (B6) and B10RIII mice using a combination of approaches, including limiting dilution assay. High numbers of in vivo primed IL-17(+) interphotoreceptor retinoid-binding protein (IRBP)-specific T cells were found in mice immunized with a uveitogenic peptide emulsified in CFA, but not the same peptide emulsified in IFA. Both in vitro and in vivo, at least part of the effect of mycobacterial antigen in CFA could be replaced by TLR2 or TLR4 ligands. TCR-δ(-/-) mice immunized with IRBP peptide in CFA generated significantly lower numbers of IL-17(+) T cells than immunized wild-type B6 mice. Administration of a small number of activated γδ T cells to TCR-δ(-/-) mice significantly increased the number of IL-17(+), but not IFN-γ(+), IRBP-specific T cells in these mice. γδ T cells from CFA- or IFA plus TLR ligand-treated mice were activated and injection of naïve TCR-δ(-/-) mice with γδ T cells from TLR ligand-treated, but not untreated, B6 mice promoted the in vivo priming of IL-17(+) IRBP-reactive T cells. In conclusion, in vivo priming of IL-17(+) uveitogenic T cells in mice is significantly affected by TLR ligation, and is also influenced by activated γδ T cells.