This study investigated whether ablation of all Toll-like receptor (TLR) signaling influenced skin allograft rejection across a complete donor/recipient mismatch of major histocompatibility and minor antigens. We predicted that defective TLR signaling would interfere with the activation of donor dendritic cells (DC) in vivo, by preventing DC activation in response to local environmental ("danger") signals. The ablation of TLR signals would therefore be associated with decreased activation of host T cells and decreased graft rejection. Using mice with deletions of the proximal TLR adapter proteins MyD88 or Trif, and those with simultaneous deletions of both MyD88 and Trif, we demonstrated that absence of both MyD88 and Trif adapter proteins prolonged skin graft survival, notably across a complete MHC and minor antigen barrier. Absence of MyD88 or Trif alone only had a modest effect on graft survival across even a minor MHC antigen difference. Prolonged survival of skin grafts from mice deficient in both MyD88 and Trif was associated with diminished migration of donor cells to draining lymph nodes and, subsequently, with delayed infiltration of host T cells into the grafted tissue.