The isolation of lymphocytes and other hematopoietic‐derived cells from small intestinal tissues has become increasingly relevant to immunology over the last decade. It is also becoming increasingly clear that the impact of local immunity at the mucosal barrier of the intestine has a profound impact on immune responses at distant sites, bringing a new cadre of immunologists to the mucosal frontier. Furthermore, the ability to experimentally manipulate smaller and smaller populations of immune cells has become technologically feasible and in some cases routine. The expanding importance of mucosal immunology coupled with increased technical capabilities requires a standard for experimentally obtaining uniform and consistent cells from the intestinal mucosa. Therefore, it is important to isolate immune cells that are highly viable and minimally manipulated to maximize cellular yields while maintaining acceptable time constraints. Curr. Protoc. Immunol. 99:3.19.1‐3.19.11. © 2012 by John Wiley & Sons, Inc.