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In some cancers and chronic infections exhausted T cells increase their expression of inhibitory receptors and demonstrate an impaired ability to produce cytokines and to proliferate. Immunological techniques such as MHC class I tetramer staining, intracellular cytokine staining, and CFSE dilution can be used to determine the memory status, inhibitory receptor expression, cytokine production, and proliferative capacity of antigen-specific CD8 + T cells. Here, we describe approaches to define the inhibitory receptor expression, cytokine production, and proliferative capacity of antigen-specific CD8 + T cells from HIV-infected and CMV-infected donors by using polychromatic flow cytometry.