Background/Objectives: Our study was designed to explore the potential role of allogeneic CD8⁺ T lymphocytes present in red blood cell concentrates (RBCs) in the development of transfusion-related immunomodulation (TRIM) and the effect of storage time on these cells. Methods: From six units of whole blood, donated by volunteers, RBCs were obtained and each one was divided into three equal parts to provide the samples for storage days 0, 21, and 42. On related days, mononuclear cells (MNCs) were isolated from these RBC samples. MNCs were cultured, and phytohemagglutinin was added to half of the culture wells to stimulate the cells and achieve T cell division. Supernatants and MNCs were obtained from stimulated (STI) and unstimulated (US) wells. Supernatants were used for cytokine analyses, while MNCs were used to investigate the T cells and transcription factors. Results: The frequency of CD8⁺ T lymphocytes (Tc), their subgroups (Tc1, Tc2, and Tc17), specific transcription factors, and effector cytokines decreased during the storage time, but cell viability increased. CD3⁺CD8⁺TNF-α+ cells were significantly higher in the STI group on day 0 compared to the US group. Other cells did not respond to the mitogen (phytohemagglutinin) stimulation. Conclusions: During storage, the number of Tc cells and their ability to respond to mitogens decreased over time. The unresponsiveness was not recovered in ex vivo cell culture. Our findings suggest that transfused Tc cells are unlikely to be primary mediators of TRIM.