Significance Cytotoxic CD8 ⁺ T cells control infectious diseases as well as cancer. Memory T cells are a subset of CD8 ⁺ T cells that have previously encountered and rapidly responded to a specific antigen. Here we identified the epigenetic writer DOT1L as a central regulator of T cell physiology. In the absence of DOT1L, which methylates histone H3K79 at active genes, cytotoxic T cells prematurely acquired memory features without having encountered an antigen. However, loss of DOT1L in the T cell lineage led to a compromised immune response. Our findings highlight the importance of epigenetic signals in regulating T cell differentiation. Understanding these epigenetic signals is important for developing strategies to modulate T cell immunity.