CD8+ T cells are important for host protection from infections and malignancies, and many current immunotherapies target molecules that enhance CD8+ T cell function and differentiation. Although genome organization is known to be important for regulating cell development and function, the changes in spatial chromatin organization and how these impact accompanying effector and memory CD8+ T cell differentiation remain unknown. Here, we studied how genome organization is integrated with other molecular mechanisms regulating CD8+ T cell differentiation and targeted CTCF, a key factor that regulates genome organization through blocking or facilitating chromatin interactions, to determine how altering interactions affect the CD8+ T cell response. We observed T cell subset-specific changes in intra-TAD interactions at sites related to transcriptional rewiring such as genes encoding for transcription factors that regulate CD8+ T cell differentiation. We next characterized the binding profile of CTCF, a known regulator of chromatin interactions. CTCF binding changed with CD8+ T cell differentiation, and weak-affinity CTCF binding is needed to promote terminal differentiation in both an infection and tumor setting through regulation of transcription programs and transcription factor activity. Strikingly, disruption of a single CTCF binding site upregulated expression of corresponding memory-associated molecules. Thus, our data show that in addition to the role for CTCF in establishing genome architecture, it also regulates the effector CD8+ T cell response through altering interactions that regulate the transcription factor landscape and transcriptome.