A consequence of naive CD8+ T cell activation is specific changes in phenotype, proliferation and acquisition of lineage-specific function. The precise gene regulatory mechanisms that control these changes are largely unknown. Using a combination of genome wide sequencing approaches we have examined the wholesale changes in both 3D structure and biochemical modifications associated with virus-specific CD8+ T cell differentiation in response to infection. Our data suggests that lineage-specific fate determination is largely preconfigured, or poised, within mature naive virus-specific CD8+ T cells. More importantly our data suggest that effector differentiation is in fact actively restrained within the naive state by specific molecular mechanisms, with T cell activation resulting in release of this molecular handbrake that triggers transcriptional activation of a highly regulated differentiation program that underpins induction of an optimal effector killer T cell response. Understanding these mechanisms is key for understanding not only how optimal CD8+ T cell effector function is established but it has implications for our understanding of how immunological memory is established, and how we may modulate this process for therapeutic gain.