Abstract Gut-enriched Krüppel-like factor (GKLF) is a transcriptional regulator expressed in differentiated epithelia. We identified GKLF transcript as a regulated element in thymic epithelium of recombinase-deficient mice during thymus development induced by anti-CD3 antibody injection. This treatment recapitulates the organogenetic process depending on productive rearrangement of T cell receptor (TCR) β gene with thymocytes expansion and acquisition of the CD4 +8 + double positive phenotype. In wildtype mice, GKLF is expressed very early in embryogenesis and becomes intensely up-regulated in thymus epithelium at day 18 of gestation when TCR β expressing cells have selectively expanded and express both CD4 and CD8. The results presented here suggest that thymocytes may regulate GKLF transcriptionally in the cortical epithelium at the developmental check-point controlled by TCR β gene rearrangement. Furthermore, GKLF expression in hematopoietic stroma might suggest the thus far uncharacterised participation of this factor in hematopoiesis.