Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. These genes are of interest in understanding immune regulation and the molecular basis of lymphoma. The present study was undertaken to characterize the early T-cell gene response in Bullmastiffs, a breed that has an increased incidence of lymphoma. Gene expression profiles were charaterized using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR), and paired samples from eleven Bullmastiff dogs. Significant functional annotation clusters were identified following stimulation with a low mitogenic dose of phytohemagluttinin (PHA) (5μg/ml), including the Toll-like receptor signalling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to cancer development, or proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2), early growth response 1 (EGR1), growth arrest and DNA damage-inducible gene (GADD45B), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS), early growth response 2 (EGR2), hemogen (HEMGN), polo-like kinase 2 (PLK2) and polo-like kinase 3 (PLK3). Differential gene expression was re-examined using qRT-PCR, which confirmed the expression patterns seen in these genes. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in cell cycle regulation and are known to be associated with cancer development in mice and humans. This study provides a comprehensive analysis of the early T-cell gene response to activation in Bullmastiffs. Key genes identified in this analysis may provide quantitative markers of T-cell response and with further study may point to underlying mechanisms of lymphoma susceptibility in this breed.