Assays to detect cytokine production and degranulation are useful measures of T cell function. Multiparametric flow cytometry makes it possible to precisely phenotype and simultaneously detect these intermediate stages of T cell activation.1,2 The degranulation of cytotoxic cells, an indicator of cytotoxic potential, results in the transient surface expression of CD107a and CD107b (lysosomal associated membrane proteins).3 A protocol for the ex vivo assessment of antigen-specific cytokine production by mouse CD4+ and CD8+ T lymphocytes, and the simultaneous presence of CD107a and CD107b, by polychromatic flow cytometry is presented here. In this protocol, C57BL/6 or Balb/c mice were immunized with a live-attenuated strain of Listeria monocytogenes (actA-Lm). Splenocytes were restimulated in vitro with peptide from listeriolysin (LLO91-99 for CD8+ (Balb/c), LLO190-201 for CD4+ (C57BL/6)) or PMA plus ionomycin. A typical surface staining cocktail consisted of CD4-APC, and CD8-PerCP-CY5.5 and a typical intracellular staining cocktail consisted of IFN-γ-PE and TNF-α-PE-Cy7. To obtain optimal surface staining for CD107a and CD107b (FITC conjugates) the antibodies were added for the entire activation period. Staining during activation allows access of the antibody to transiently expressed CD107a and CD107b antigens.