RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family with unknown physiological function. Here we demonstrate that Rhoh^−/− mice have impaired T cell receptor (TCR)–mediated thymocyte selection and maturation, resulting in T cell deficiency. RhoH deficiency resulted in defective CD3ζ phosphorylation, impaired translocation of the signaling molecule Zap70 to the immunological synapse and reduced activation of Zap70-mediated signaling in thymic and peripheral T cells. Proteomic analyses demonstrated that RhoH is a component of TCR signaling and is required for recruitment of Zap70 to the TCR through interaction with RhoH noncanonical immunoreceptor tyrosine-based activation motifs (ITAMs). In vivo reconstitution studies also demonstrated that RhoH function depends on phosphorylation of the RhoH ITAMs. These findings suggest that RhoH is a critical regulator of thymocyte development and TCR signaling by mediating recruitment and activation of Zap70.