Wt1 is specifically expressed in Sertoli cells in the developing testis. A previous study has demonstrated that Wt1 plays a critical role in maintaining the integrity of testicular cords. However, the underlying mechanism is unclear. In this study, we found that the laminin-positive basal lamina lining the testicular cords was fragmented and completely absent in some areas of Wt1^−/flox; Amh-Cre testes, indicating that the testicular cord disruption can be attributed to the breakdown of the basement membrane. To explore the molecular mechanism underlying this effect, we examined the expression of cell adhesion molecules (CAMs) and testicular cord basal lamina components by real-time RT-PCR, Western blotting, and immunostaining. Compared with control testes, the expression of CAMs (such as E-cadherin, N-cadherin, claudin11, occludin, beta-catenin, and ZO-1) was not obviously altered in Wt1^−/flox; Amh-Cre testes. However, the mRNA level of Col4a1 and Col4a2 was significantly decreased in Wt1-deficient testes. Immunostaining assays further confirmed that the collagen IV protein levels were dramatically reduced in Wt1^−/flox; Amh-Cre testes. Moreover, luciferase and point mutation analyses revealed that the Col4a1 and Col4a2 promoters were additively transactivated by WT1 and SOX9. Given this finding and previous results showing that SOX9 expression declines rapidly after Wt1 deletion, we conclude that the loss of Wt1 in Sertoli cells results in the downregulation of the important basal lamina component, which in turn causes the breakdown of the basal lamina and subsequent testicular cord disruption.