The intestinal tissue is charged with a delicate immunological task. The intestinal immune system needs to be tolerant towards nutrients and microbiota present in the intestinal lumen, while simultaneously detecting and responding to dangers such as pathogens. A single-cell layer of intestinal epithelial cells (IECs) acts as a first line of defence. There is a T cell population located between the IECs that have been named intraepithelial lymphocytes (IELs). As the main lymphoid population within the intestinal barrier, IELs are thought to have an important role in intestinal homeostasis maintenance, as well as a role in intestinal inflammatory and autoimmune diseases such as inflammatory bowel disease and celiac disease. Despite extensive research on IEL biology, there are still questions remaining in terms of the development, maintenance and activation of IELs. Furthermore, IELs survive poorly in vitro, which hinders mechanistic insights. In this thesis, a co-culture system between IELs and intestinal organoids, “mini-guts”, provides an in vitro model for IELs. With this IEL-organoid co-culture system, IELs associated with the organoids survive for at least 4 days. Additional findings suggest that IELs are kept in a poised state of activation due to differences in their mitochondria compared to other T cells found in spleen, lung and skin. Upon activation or intestinal inflammation, the mitochondrial mass in IELs increases. This increase correlates with effector functions such as cytokine production and proliferation. In addition, the composition of the mitochondria-specific lipid, cardiolipins, alters drastically in IELs after activation. These data support a model of mitochondria-dependent activation of IELs. The mitochondria-dependent activation in IELs appears to have at least two pathways: one T cell receptor-dependent and one microbiota-dependent. The latter pathway suggests a model in which IELs can become activated regardless of the cause of intestinal epithelial barrier damage.