Mobilization of tumor-reactive CD8+ T cells remains the major challenge of cancer immunotherapy. Knowing how and when the T cell response expands and differentiates after antigen stimulation would make a significant contribution to the development of tumor vaccines. In the current study, we used CFSE-based cell sorting and cDNA microarray to identify the gene expression profile of adjacent generations of T cells after PHA stimulation. Early-divided generations of T cells responded to stimulation by activating cell cycle and surviving gene pathways, while late generations of T cells had more dramatic changes in transcription of cytokine genes. Reconstruction of biochemical pathways, activated in both early and late generations of T cells, also confirmed the impact of division in focal-adhesion kinases. Because most tumors are infiltrated by lymphocytes, our studies indicate a novel approach to identify 'systemic biological responses' of T cells, which could determine the design, and optimization of effective tumor vaccines.