Autoimmune regulator (Aire) is essential in the perinatal period to prevent the multiorgan autoimmunity. Here we show that Aire-regulated single positive thymocyte trafficking in neonatal period is critical for thymic egress. Reduced thymic emigration was found in Aire(-/-) mice during neonatal period, leading to enhanced homeostatic expansion of peripheral T cells as early as 2 weeks of age. In neonatal Aire(-/-) mice, thymic expression of CCR7 ligands were dramatically reduced, resulting in decreased thymocyte motility and thymocyte emigration. This reduction of thymic egress in Aire(-/-) mice was alleviated beyond 3 weeks of age by an early upregulation of S1P(1) signaling. As the numbers and quality of thymic emigrants are essential for the establishment and maintenance of peripheral tolerance, the reduced thymic emigration during neonatal period may deteriorate autoimmunity caused by the emigration of autoreactive T cells.