Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTβR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTβR expression was found in all TEC subsets, but the highest levels were detected in MTS-15+ thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTβR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEClow), as well as maintenance of MTS-15+ fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta–/– and Ltbr–/– mice. Thus, we propose a broader role for LT1β2-LTβR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEClow for efficient induction of central tolerance. ; Natalie Seach, Tomoo Ueno, Anne L. Fletcher, Tamara Lowen, Monika Mattesich, Christian R. Engwerda, Hamish S. Scott, Carl F. Ware, Ann P. Chidgey, Daniel H. D. Gray and Richard L. Boyd ; Copyright © 2008 by The American Association of Immunologists, Inc.