We determined how CD8+ melanoma tumor–infiltrating lymphocytes (TILs) isolated from two distinct phases of expansion in preparation for adoptive T cell therapy respond to melanoma Ag restimulation. We found that TILs isolated after the rapid expansion protocol (REP) phase, used to generate the final patient TIL infusion product, were hyporesponsive to restimulation with MART-1 peptide-pulsed dendritic cells, with many CD8+ T cells undergoing apoptosis. Telomere length was shorter post-REP, but of sufficient length to support further cell division. Phenotypic analysis revealed that cell-surface CD28 expression was significantly reduced in post-REP TILs, whereas CD27 levels remained unchanged. Tracking post-REP TIL proliferation by CFSE dilution, as well as sorting for CD8+ CD28+ and CD8+ CD28− post-REP subsets, revealed that the few CD28+ TILs remaining post-REP had superior survival capacity and proliferated after restimulation with MART-1 peptide. An analysis of different supportive cytokine mixtures during the REP found that a combination of IL-15 and IL-21 facilitated comparable expansion of CD8+ TILs as IL-2, but prevented the loss of CD28 expression with improved responsiveness to antigenic restimulation post-REP. These results suggest that current expansion protocols using IL-2 for melanoma adoptive T cell therapy yields largely CD8+ T cells unable to persist and divide in vivo following Ag contact. The few CD8+ CD28+ T cells that remain may be the only CD8+ TILs that ultimately survive to repopulate the host and mediate long-term tumor control. A REP protocol using IL-15 and IL-21 may greatly increase the number of CD28+ TILs capable of long-term persistence.