Gene deletion experiments have shown that the three TGFbeta isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins alphavbeta6 and alphavbeta8 activate latent forms of TGFbeta1 and TGFbeta3. This raises the possibility that TGFbeta1 and TGFbeta3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFbeta1 activation (Tgfb1(RGE/RGE)) that were also homozygous for a null mutation in the TGFbeta3 gene. Tgfb1(RGE/RGE); Tgfb3(-/-) mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking alphavbeta8. Some Tgfb1(RGE/RGE); Tgfb3(+/-) and Tgfb1(RGE/RGE); Tgfb3(+/+) mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFbeta1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3(-/-) mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFbeta1 and TGFbeta3 during development based on a shared mechanism of activation.