Cryptopatches (CP) are murine gut anatomical sites for generating thymus-independent intraepithelial T lymphocytes (IEL). However, it remains elusive how lympho-hematopoietic progenitor cells migrate from bone marrow (BM) into CP and differentiate into IEL. Here we show that mice reconstituted with BM-derived c-kit(+) cells express CCL25 (TECK)-intrakine gene, which reduces specifically the chemotactic response to CCL25 but not CXCL12 in the thymocytes. These mice exhibited a dramatic reduction of CP and IEL in the small intestine, and harbored conspicuously decreased numbers of c-kit(+) cells in the emaciated CP. In contrast, T cells in the thymic, splenic and lymph node compartments developed normally in these mice. Importantly, it was demonstrated that CD11c(+) dendritic stromal cells in CP expressed CCL25 and c-kit(+) Lin(-) BM cells displayed vigorous chemotactic response to CCL25. Furthermore, RT-PCR analysis detects mRNA expression of CCR9 in the c-kit(+) Lin(-) BM cells. Thus, these results demonstrate that the CCL25-CCR9 pathway is essential for CP formation and the consequent appearance of IEL.