Natural killer T (NKT) cells rapidly respond to antigenic stimulation with cytokine production and direct cytotoxicity. These innate-like characteristics arise from their differentiation into mature effector cells during thymic development. A subset of mature NKT cells remain thymic resident, but their activation and function remain poorly understood. We examined the roles of CD28 and CTLA-4 in driving the activation of thymic resident NKT cells. In contrast to studies with peripheral NKT cells, the proliferation of thymic NKT cells was significantly impaired when CD28 engagement was blocked, but unaffected by CTLA-4 activation or blockade. Within NKT subsets, however, stage 3 NKT cells, marked by higher NK1.1 expression, were significantly more sensitive to the loss of CD28 signals compared to NK1.1- stage 2 NKT cells. In good agreement, CD28 blockade suppressed NKT cell cytokine secretion, lowering the ratio of IFN-γ:IL-4 production by NK1.1+ NKT cells. Intriguingly, the activation-dependent upregulation of the master transcription factor PLZF did not require CD28-costimulation in either of the thymic NKT subsets, underlining a dichotomy between requirements for early activation vs subsequent proliferation and effector function by these cells. Collectively, our studies demonstrate the ability of CD28 co-stimulation to fine tune subset-specific responses by thymic resident NKT cells and contextually shape the milieu in this primary lymphoid organ.