Mice of the B10 background that are class II I-E nonexpressing demonstrate a relative resistance to neonatal induction of tolerance of class I alloantigens from I-E-expressing B10 strains. The majority of these injected mice delete donor-responsive and “I-E-reactive” (Vβ11+) cells in the immediate postinoculation period, with many remaining deleted of donor-specific T cells before the application of the test skin graft. Utilizing a hemisplenectomy technique in B10.S (I-E−) mice that received as neonates MHC-disparate (B10.S±B10.A)F1 (I-E+) lymphohematopoietic cells, we determined the proportion of adult mice that demonstrated pretrans-plant donor cell chimerism as well as several functional and phenotypic features ascribed to donor responsiveness. Surprisingly, chimeric cells were present in the bulk of recipients and many also exhibited a deletion of Vβ11+ T cells and a lack of alloreactivity to the donor strain allotype. Since chimeric, MLR−, Vβ11-deleted mice would be predicted to be tolerant to allograft challenge, we hypothesized that the test skin graft applied in adulthood was providing stimulatory signals that overcame this state of immunologic unresponsiveness. To examine this issue, injected mice that had been evaluated before skin grafting were challenged with donor-specific skin and evaluated for the same parameters measured in the pretransplant period. The majority (95%) of these mice subsequently rejected the B10.A skin graft in a range of 7–14 days. After graft rejection, Vβ11= cell levels generally increased and chimeric cells were typically eliminated. Thus, the ability to reject allografts is not predicted by a “nonresponsive” immune phenotype or the presence of chimeric cells before application of the test allograft. In fact, the graft appears to provide an in vivo stimulus to the reduced numbers of host donor-specific T cells that results in the removal of chimeric cells and a breakdown of the tolerant state. We conclude that application of orthotopic skin grafts provides the signal(s) necessary to break class I tolerance induced neonatally in the context of I-E disparity.