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Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) use cytotoxic granules containing perforin and granzymes to lyse infected or malignant host cells, thereby providing immunity to intracellular microbes and tumors. Perforin is essential for cytotoxic granule‐mediated killing. Perforin expression is regulated transcriptionally and correlates tightly with the development of cells that can exhibit cytotoxic activity. Although a number of genes transcribed by T cells and NK cells have been studied, the cell‐specificity of perforin gene expression makes it an ideal model system in which to clarify the transcriptional mechanisms that guide the development and activation of cytotoxic lymphocytes. In this review, we discuss what is known about perforin expression and its regulation, then elaborate on recent studies that utilized chromosome transfer and bacterial artificial chromosome transgenics to define a comprehensive …