Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through release of perforin-containing lytic granules. Here, we first performed probability state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTL with NK cells. Analysis identified CD57(bright)-expression as a reliable phenotype of granule marker-containing CTL. We then compared CD3(+)CD8(+)CD57(bright) CTL with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTL expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. Upon stimulation, such CTL degranulated more readily than other T cell subsets, but had a similar propensity to degranulate as NK cells. Remarkably, the CTL produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistocytosis (FHL), CTL and NK cell degranulation was similarly impaired. Thus, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTL and NK cells, and suggest that analysis of CD57(bright) CTL-function may prove useful in the diagnosis of primary immunodeficiencies including FHL.