A population of CD8+ CTL can be generated in vitro in the presence of anti-CD8 mAb. Due to their apparent high avidity characteristic, these anti-CD8-resistant CD8+ CTL may have important functional in vivo roles in graft rejection, and may be important in antiviral and antitumor responses. We have previously reported that this anti-CD8-resistant subset of CD8+ CTL demonstrates functional differences from anti-CD8-sensitive CD8+ CTL. One important difference between the subsets is the markedly greater dependence of anti-CD8-resistant CTL upon exogenous cytokines for their generation in vitro. In this study, we have investigated in detail the cytokine requirements for the generation of allospecific CD8+ CTL in vitro and have found that IL-4 can augment the generation of anti-CD8-sensitive but not anti-CD8-resistant CTL, whereas IL-2 or IL-12 can augment the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL. However, anti-CD8-resistant CTL require at least 10-fold higher concentrations of IL-2 than do anti-CD8-sensitive CTL. This more stringent IL-2 requirement precludes the efficient generation of anti-CD8-resistant CTL in vitro in the absence of exogenous IL-2 because they cannot produce sufficient IL-2 to meet their needs, in contrast to anti-CD8-sensitive CTL. By providing exogenous cytokines to allospecific CTL generation cultures, we further demonstrate that anti-CD8-resistant CTL can be functionally skewed to the Tc1 subset, but differ from anti-CD8-sensitive conventional CTL in that they cannot be skewed to the Tc2 subset.