Viral infection triggers proliferation and differentiation of naïve, quiescent CD8+ T cells, resulting in a large pool of effector cells capable of killing infected host cells. Importantly, infection gives rise to long-lived virus-specific memory T cells that reactivate rapidly following re-infection, providing the basis of T cell-mediated immunity. While it is understood that T cell differentiation is orchestrated by global changes in gene transcription, the mechanisms that result in these changes are unknown. Global changes in nuclear architecture occur following T cell activation, with modulated deposition of histone modifications and locus-specific regulatory interactions acting to fine-tune gene transcription. At a broader level, the positioning of genes within the nucleus is regulated to influence expression by altering proximity to transcriptional machinery, while higher-order chromatin structures have an uncharacterised role in imparting cell-type specific transcription profiles. The question of how these different layers of regulation work together to choreograph differentiation outcomes has only recently been addressed. Here we report application of single molecule localization microscopy (dSTORM) combined with genomics techniques to determine how higher order chromatin structures are modulated to regulate T cell differentiation, and how these structural changes control gene transcription to facilitate anti-viral immunity.