The onset of protective immunity against pathogenic SIV challenge in SIVnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in nave and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized nave and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from nave animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states.