How organ-specific central tolerance is established and regulated has been an intriguing question. Lymphotoxin beta receptor (LTbetaR) deficiency is associated with autoimmune phenotypes characterized by humoral and cellular autoreactivity to peripheral organs. Whether this results from defective negative selection of T cells directed at tissue-restricted Ags has not been well understood. By tracing the development of OT-I thymocytes in rat insulin 2 promoter-mOVA transgenic mice on either Ltbr+/+ or Ltbr-/- background, we demonstrate that LTbetaR is necessary for thymic negative selection. LTbetaR deficiency resulted in a dramatic escape of "neo-self" specific OT-I cells that persist in circulation and lead to development of peri-insulitis. When the underlying mechanism was further explored, we found interestingly that LTbetaR deficiency did not result in reduced thymic expression of mOVA. Instead, LTbetaR was revealed to control the expression of thymic medullary chemokines (secondary lymphoid tissue chemokine (SLC) and EBV-induced molecule 1 ligand chemokine (ELC)) which are required for thymocytes migration and selection in medulla. Furthermore, RIP-mOVA transgenic mice on SLC/ELC deficient background (plt) demonstrated significant impaired negative selection of OT-I cells, suggesting that the dysregulation of SLC/ELC- expression alone in Ltbr-/- thymi can be sufficient to impair thymic negative selection. Thus, LTbetaR has been revealed to play an important role in thymic negative selection of organ-specific thymocytes through thymic medullary chemokines regulation.