The T cell compartment is phenotypically and functionally heterogeneous; subsets of naive and memory cells have different functional properties, and also differ with respect to homeostatic potential and the ability to persist in vivo. Human stem cell memory T (T_SCM) cells, which possess superior immune reconstitution and antitumor response capabilities, can be identified by polychromatic flow cytometry on the basis of the simultaneous expression of several naive markers together with the memory marker CD95. We describe here a protocol based on the minimum set of markers required for optimal identification of human and nonhuman primate (NHP) T_SCM cells with commonly available flow cytometers. By using flow sorters, T_SCM cells can thereby be isolated efficiently at high yield and purity. With the use of the 5.5-h isolation procedure, depending on the number of cells needed, the sorting procedure can last for 2–15 h. We also indicate multiple strategies for their efficient expansion in vitro at consistent numbers for functional characterization or adoptive transfer experiments.