IL-7 is a cytokine crucial for development and maintenance of lymphocytes and other hematopoietic cells. However, how IL-7-expressing cells are distributed in lymphoid organs is not well known. To address this question, we established and analyzed IL-7-GFP knock-in mice. Thymic epithelial cells (TECs) expressed high GFP levels in the cortex and medulla, as detected with an anti-GFP Ab. Thymic mesenchymal cells also expressed GFP. Flow cytometry analysis suggested that cortical TECs expressed higher GFP levels than did medullary TECs. In bone marrow, immunohistochemistry indicated high levels of GFP in many VCAM-1(+) mesenchymal stromal cells and in some VCAM-1(-) cells. Additionally, half of the VCAM-1(+)CD31(-) stromal cells and some platelet-derived growth factor receptor alpha(+) stromal cells were GFP(+), as detected by flow cytometry. Moreover, we detected GFP expression in fibroblastic reticular cells in the T cell zone and cortical ridge of lymph nodes. Remarkably, lymphatic endothelial cells (LECs) expressed GFP at high levels within the lymph node medulla, skin epidermis, and intestinal tissues. Additionally, we detected abundant IL-7 transcripts in isolated LECs, suggesting that LECs produce IL-7, a heretofore unknown finding. Furthermore, GFP is expressed in a subpopulation of intestinal epithelial cells, and that expression was markedly upregulated in a dextran sulfate sodium-induced acute colitis model. Overall, IL-7-GFP knock-in mice serve as a unique and powerful tool to examine the identity and distribution of IL-7-expressing cells in vivo. The Journal of Immunology, 2012, 189: 1577-1584.