Tissue-resident memory T cells are critical mediators of viral and tumor immunity and are increasingly being recognized as key players in autoimmune and allergic pathologies. Despite this, targeting tissue-resident lymphocytes specifically is currently difficult, as their developmental requirements are only partially understood. Using sophisticated genetic and molecular approaches in combination with animal models, we have identified common genes and molecular pathways that drive the tissue-resident cell fate, as well as tissue-specific factors that imprint distinct gene signatures on immune cells that reside in different microenvironments. Combined, this work provides a molecular framework for tissue-resident lymphocyte differentiation, revealing novel pathways that may be targeted therapeutically.