Crosstalk between immune cells and intestinal stem cells (ISCs) in vivoplays a critical role in tissue homeostasis and inflammation; however, in vitromodels based on primary cells recapitulating this interaction were lacking. Here, we provide a detailed protocol for an autologous in vitrolong-term 3D co-culture system of human intestinal CD4+T cells and ISCs to study T cell-intestinal epithelial cell interactions during tissue development and inflammation.