The study of T cell biology has been accelerated by substantial progress at the technological level, particularly through the continuing advancement of flow cytometry. The conventional approach of observing T cells as either T helper or T cytotoxic is overly simplistic and does not allow investigators to clearly identify immune mechanisms or alterations in physiological processes that impact on clinical outcomes. The complexity of T cell sub‐populations, as we understand them today, combined with the immunological and functional diversity of these subsets represent significant complications for the study of T cell biology. In this article, we review the use of classical markers in delineating T cell sub‐populations, from “truly naïve” T cells (recent thymic emigrants with no proliferative history) to “exhausted senescent” T cells (poorly proliferative cells that display severe functional abnormalities) wherein the different phenotypes of these populations reflect their disparate functionalities. In addition, since persistent infections and chronological aging have been shown to be associated with significant alterations in human T cell distribution and function, we also discuss age‐associated and cytomegalovirus‐driven alterations in the expression of key subset markers. © 2013 International Society for Advancement of Cytometry