The activation state of alloreactive T cells correlates with skin allograft outcome in mice treated with costimulation blockade. C57BL/6 mice were treated with anti-CD154 mAb and a BALB/c DST and engrafted with BALB/c skin as described in “Materials and methods.” At 101 days after transplantation, (A) 4 of 12 C57BL/6 mice retained intact BALB/c skin grafts and (B) 8 of 12 mice had completely rejected the allogeneic skin or were in the process of graft rejection. Splenocytes were harvested from the recipient mice 101 days after skin transplantation and stimulated in vitro with syngeneic C57BL/6 (H2^b) or allogeneic BALB/c (H2^d) splenocytes. Samples were then stained for cell surface markers and for TNF-α or IFN-γ. Samples were gated on CD8⁺ cells, and the values shown represent the percentage of CD8⁺ T cells staining positive for TNF-α or IFN-γ. Representative dot plots are shown.