Innate and adaptive cellular immunity is initiated, directed and regulated by a vast array of cell surface receptors. Attempts to harness the cellular immune system in translational settings such as immunotherapy and vaccine development require tools to accurately describe and isolate lymphocytes with specific characteristics. One such tool, flow cytometry, is undergoing a revolution in instrumentation and reagents, providing opportunities for high resolution phenotypic and functional analysis of lymphocytes. Here, we demonstrate how nine-color flow cytometry can be adapted, optimized and applied to investigate the phenotypic complexity and functional heterogeneity of human lymphocyte subsets. We provide examples of studies of adaptive T cell responses against viruses, as well as the assessment of CD1d-restricted NKT cells and NK cells. We discuss the importance of this technology for detailed investigations of lymphocyte subsets in studies of infectious diseases and cancer.