Although the importance of genome organization for transcriptional regulation of cell-fate decisions and function is clear, the changes in chromatin architecture and how these impact effector and memory CD8+ T cell differentiation remain unknown. Using Hi-C, we studied how genome configuration is integrated with CD8+ T cell differentiation during infection and investigated the role of CTCF, a key chromatin remodeler, in modulating CD8+ T cell fates through CTCF knockdown approaches and perturbation of specific CTCF-binding sites. We observed subset-specific changes in chromatin organization and CTCF binding and revealed that weak-affinity CTCF binding promotes terminal differentiation of CD8+ T cells through the regulation of transcriptional programs. Further, patients with de novo CTCF mutations had reduced expression of the terminal-effector genes in peripheral blood lymphocytes. Therefore, in addition to establishing genome architecture, CTCF regulates effector CD8+ T cell heterogeneity through altering interactions that regulate the transcription factor landscape and transcriptome. Display omitted •Genome organization changes with effector CD8+ T cell differentiation•CTCF-binding patterns are altered with effector CD8+ T cell differentiation•CTCF regulates subset-specific transcriptional programs and chromatin accessibility•CTCF controls optimal CD8+ T cell-fate decisions in response to infection How changes in spatial chromatin organization are integrated into the network of molecular mechanisms mediating CD8+ T cell effector functions and memory formation is not well understood. Quon et al. characterize genome interactions accompanying the CD8+ T cell response to infection and find that the DNA architectural protein, CTCF, regulates the balance of terminally differentiated-effector cells and memory-fated cells.