BACKGROUND: The induction of tolerance toward third-party solid organ grafts with allogeneic thymus tissue transplantation has not been previously demonstrated in human subjects. OBJECTIVE: Infants with complete DiGeorge anomaly (having neither thymus nor parathyroid function) were studied for conditions and mechanisms required for the development of tolerance to third-party solid organ tissues. METHODS: Four infants who met the criteria received parental parathyroid with allogeneic thymus transplantation and were studied. RESULTS: Two of 3 survivors showed function of both grafts but subsequently lost parathyroid function. They demonstrated alloreactivity against the parathyroid donor in mixed lymphocyte cultures. For these 2 recipients, parathyroid donor HLA class II alleles were mismatched with the recipient and thymus. MHC class II tetramers confirmed the presence of recipient CD4(+) T cells with specificity toward a mismatched parathyroid donor class II allele. The third survivor has persistent graft function and lacks alloreactivity toward the parathyroid donor. All parathyroid donor class II alleles were shared with either the recipient or the thymus graft, with minor differences between the parathyroid (HLA-DRB1∗1104) and thymus (HLA-DRB1∗1101). Tetramer analyses detected recipient T cells specific for the parathyroid HLA-DRB1∗1104 allele. Alloreactivity toward the parathyroid donor was restored with low doses of IL-2. CONCLUSION: Tolerance toward parathyroid grafts in combined parental parathyroid and allogeneic thymus transplantation requires matching of thymus tissue to parathyroid HLA class II alleles to promote negative selection and suppression of recipient T cells that have alloreactivity toward the parathyroid grafts. This matching strategy may be applied toward tolerance induction in future combined thymus and solid organ transplantation efforts.