Abstract TCR reactivity is tuned during thymic development. Immature thymocytes respond to low-affinity self-ligands resulting in positive selection. Following differentiation, T cells no longer respond to low-affinity ligands, but respond well to high-affinity (foreign) ligands. We show in this study that this response includes integrin activation, supramolecular activation cluster formation, Ca2+ flux, and CD69 expression. Because glycosylation patterns are known to change during T cell development, we tested whether alterations in sialylation influence CD8 T cell sensitivity to low affinity TCR ligands. Using neuraminidase treatment or genetic deficiency in the ST3Gal-I sialyltransferase, we show that desialylation of mature CD8 T cells enhances their sensitivity to low-affinity ligands, although these treatments do not completely recapitulate the dynamic range of immature T cells. These studies identify sialylation as one of the factors that regulate CD8 T cell tuning during development.