<p>CD8⁺ T cells are rendered exhausted in cancer, accompanied by extensive changes in the epigenome. However, whether and how higher-order chromatin organization is involved in exhausted CD8⁺ T cell differentiation is unclear. Here, we show extensive changes in the three-dimensional genome during T cell exhaustion associated with changes in gene transcription. Moreover, we identified interferon regulatory factor 8 (IRF8) as an essential transcription factor involved in reorganization of the spatial proximity between enhancers and promoters of genes associated with exhausted T cell differentiation. IRF8 deficiency inhibited the differentiation of exhausted CD8⁺ T cells and their antitumor function. Mechanistically, IRF8 bound to genes associated with exhausted T cell differentiation and promoted the formation of intra-TAD chromosomal loops. At the loop anchor regions, IRF8 recruited CTCF to form active chromosomal structures to regulate gene transcription. These results thus identify a critical role of IRF8-dependent chromatin topology during exhausted CD8⁺ T cell differentiation.</p>