Background: Acute kidney injury (AKI) is a leading cause of early post-transplant kidney damage. Furthermore, acute tubular necrosis (ATN) is appointed as the most prevalent form of AKI, a frequent multifactorial process associated with high morbidity and mortality, yet giving rise to delayed graft function (DGF) and, ultimately, allograft dysfunction. Common factors such as prolonged cold ischemia time, advanced donor age, cadaveric versus living donor, donor history of hypertension, as well as donation after cardiac death have all been deemed risk factors for ATN. With the increasing number of older cadaveric and cardiac donors in the donation process, ATN could have a detrimental impact on patient welfare. Therefore understanding the underlying process would benefit the transplant outcome. We aimed to prospectively monitor several T cell subsets in a cohort of kidney transplant recipients (KTrs) to investigate whether there is an adaptive immune-mediated involvement in the ATN process. Methods: Peripheral blood was collected from 31 KTrs at different time points within the first-year post-transplantation for in vitro stimulation with Concanavalin-A (Con-A) in a humidified 5% CO{}_2 incubator at 37 °C for 72 hours. Upon cell stimulation, flow cytometry was applied to quantify the surface expression through the median fluorescence intensity (MFI) of CD4{}^{+}CD25{}^{+}, CD8{}^{+}CD25{}^{+}, CD4{}^{+}CD38{}^{+}, CD8{}^{+}CD38{}^{+}, CD4{}^{+}CD154{}^{+}, CD8{}^{+}CD154{}^{+}, CD4{}^{+}CD69{}^{+}, CD8{}^{+}CD69{}^{+}, CD4{}^{+}CD95{}^{+}, and CD8{}^{+}CD95{}^{+} T cells. Statistical analysis was carried out with SPSS Statistics IBM v.25 (IBM Corp, Armonk, NY, USA). MFIs values were compared using a univariate analysis by a nonparametric U-Mann Whitney test. ROC analysis was applied to define cut-off values most capable of stratifying patients at high risk of ATN. Spearman’s rank-order coefficient test was applied to correlate biomarkers with allograft function. Multivariate regression independently validated CD8{}^{+} T lymphocytes as surrogate biomarkers of ATN. A p-value 0.05 was considered statistically significant. Results: KTrs who developed ATN upon transplantation had significantly higher expression of CD25, CD69, and CD95 on CD8{}^{+} and lower expression of CD95 on CD4{}^{+} T lymphocytes than patients with stable graft function. ROC curve analysis showed that MFIs \ge1015.20 for CD8{}^{+}CD25{}^{+}, \ge2489.05 for CD8{}^{+}CD69{}^{+}, \ge4257.28 for CD8{}^{+}CD95{}^{+}, and \le1581.98 for CD4{}^{+}CD95{}^{+} were capable of stratifying KTrs at high risk of ATN. Furthermore, patients with an MFI below any cut-off were significantly less likely to develop ATN than those with other values. The allograft function was correlated with the CD4{}^{+}CD95{}^{+}/CD8{}^{+}CD95{}^{+} ratio in KTrs who developed ATN. The multivariate analysis confirmed that, within the first-month post-transplant, MFI values of CD8{}^{+}CD25{}^{+}, CD4{}^{+}CD95{}^{+}, and CD8{}^{+}CD95{}^{+} T lymphocytes, along with donor age, serum creatinine, and GFR were independent risk factors to ATN. Moreover, we were also able to corroborate previous immune factors of importance in immune-mediated response to the allograft, such as the patient’s maximum panel reactive antibody (PRA) or the maintenance immunosuppression therapy. Conclusions: Our results demonstrate evidence for the implication of CD8{}^{+} T lymphocytes in the development of ATN early in the post-transplant phase. Post-transplant monitoring of activated CD8{}^{+} T lymphocytes may help identify which patients require further clinical intervention to prevent graft damage.