Abstract The α4 integrins α4β7 and α4β1, and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of α4 integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express α4β7 and α4β1, including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal β7−/− mice and adult and neonatal mice given blocking Abs to α4β7, MAdCAM-1, or VCAM-1 did not identify a role for α4 integrins in cryptopatch (CP) development; however, these studies demonstrated that α4β7 and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that β7−/− LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that β7 expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for α4β7/MAdCAM-1 in ILF development corresponding to the influx of β7-expressing lymphocytes and a nonessential role for β7-localizing LTi-like cells to the small intestine.