Liver X receptors (LXRs) are known as key transcription factors in lipid metabolism and have been reported to play an important role in T-cell proliferation. However, whether LXRs play a role in thymocyte development remains largely unknown. Here, we demonstrated that LXRβ deficiency caused a reduction in single-positive (SP) thymocytes, whereas the transitions from the double-negative to SP stage were normal. Meanwhile, LXRβ-null SP thymocytes exhibited increased apoptosis and impairment of the IL-7Rα-Bcl2 axis. In addition, the LXR agonist T0901317 promoted the survival of SP thymocytes with enhanced IL-7Rα expression in wild-type mice but not in LXRβ-deficient mice. Mechanistically, LXRβ positively regulated the expression of IL-7Rα via direct binding to the Il7r allele in SP thymocytes, and forced expression of IL-7Rα or Bcl2 restored the survival of LXRβ-defective SP thymocytes. Thus, our results indicate that LXRβ functions as an important transcription factor upstream of IL-7Rα to promote the survival of SP thymocytes.