Various cosignaling molecules on T cells can contribute to activation, inhibition, or exhaustion, depending on context. The surface receptor signaling lymphocytic activation molecule (SLAM) family receptor CD244 (2B4/SLAMf4) has been shown to be capable of either inhibitory or enhancing effects upon engagement of its ligand CD48 (SLAMf2). We examined phenotypes of CD8 T cells from HIV(+) and HIV(neg) human donors, specific for HIV and/or respiratory syncytial virus. Cultured and ex vivo CD8 T cells expressed PD-1, CD244, and TIM-3. We found that ex vivo CD8 T cells downregulated CD244 in response to superantigen. Furthermore, cognate peptide induced rapid downregulation of both CD244 and TIM-3, but not PD-1, on CD8 T cell clones. CD244 downmodulation required simultaneous signaling via both TCR and CD244 itself. Using a pH-sensitive fluorophore conjugated to avidin-Ab tetramers, we found that CD244 crosslinking in the presence of TCR signaling resulted in rapid transport of CD244 to an acidic intracellular compartment. Downregulation was not induced by PMA-ionomycin, or prevented by PI3K inhibition, implicating a TCR-proximal signaling mechanism. CD244 internalization occurred within hours of TCR stimulation and required less peptide than was required to induce IFN-γ production. The degree of CD244 internalization varied among cultured CD8 T cell lines of different specificities, and correlated with the enhancement of IFN-γ production in response to CD48 blockade in HIV(+), but not HIV(neg), subjects. Our results indicate that rapid CD244 internalization is induced by a two-signal mechanism and plays a role in modulation of antiviral CD8 T cell responses by CD48-CD244 signaling.