During positive selection, thymocytes transition through a stage during which T cell antigen receptor (TCR) signaling controls CD4-versus-CD8 lineage 'choice' and subsequent maturation. Here we describe a previously unknown T cell–specific protein, Themis, that serves a distinct function during this stage. In Themis^−/− mice, thymocyte selection was impaired and the number of transitional CD4⁺CD8^int thymocytes as well as CD4⁺ or CD8⁺ single-positive thymocytes was lower. Notably, although we detected no overt TCR-proximal signaling deficiencies, Themis^−/− CD4⁺CD8^int thymocytes showed developmental defects consistent with attenuated signaling that were reversible by TCR stimulation. Our results identify Themis as a critical component of the T cell developmental program and suggest that Themis functions to sustain and/or integrate signals required for proper lineage commitment and maturation.