The acquisition of cytotoxic functions by effector CD8+ T cells (CTL) is a critical component of the adaptive immune system as they provide targeted antigenic responses to infections. This differentiation process, which requires the integration of T cell and co-stimulatory signaling cascades, is underscored by epigenetic re-programming, where coordinated changes to chromatin structure and histone modifications allow establishment of CTL transcriptional signatures. Recently, the regulation of a specific repressive histone modification (H3K27me3) has been shown to influence naïve to effector/memory differentiation in CD8+ T cells. Here, we track the dynamics of H3K27me3 demethylation during the early phase of T cell activation against Influenza A in an in vitro mouse model. Using ChIP- and RNA-sequencing, we demonstrate that early-activated T cells engage in a series of stepwise transcriptional changes, with transcription factors critical for T cell differentiation such as Tbx21 (encoding for T-bet) and Irf4 being expressed at as early as 3 hours post-activation. Importantly, the use of a demethylase inhibitor demonstrated that the magnitude of their transcription responses was dependent on H3K27me3 demethylation, indicating that the removal of this repressive histone modification significantly shapes early cytotoxic gene expression programs through the activation of these transcription factors. Understanding these molecular signatures therefore represents a novel regulatory step involved in the formation of effector and memory CD8+ T cells.