In normal conditions, body temperature is tightly maintained at approximately 37 oC. However, in case of infection, body temperature can change by increasing or decreasing its value, resulting in fever or regulated hypothermia, respectively. This change in body temperature has a role in the anti-pathogenic response, as well as in the modulation of the immune system. Little is known about how temperature affects immune cells. To investigate how temperature affects CD8+ T cells, we used CD8+ T cells isolated from OT-1 mice. CD8+ T cells were separated by MACS and stimulated with SIINFEKL peptide at concentrations of 0, 0.1, 1, and 10 ng/ml and costimulated with 0.5 μL/ml of αCD28. Cells were incubated at 34.5 oC and 37 oC for 3 days, after which proliferation, expression of activation markers and production of cytotoxic cytokines were analyzed using flow cytometry. Results showed decreased proliferation and cytokine production, as well as diminished differentiation into effector type in cells cultured at a lower temperature of 34 oC compared to cells at the physiologically optimal temperature of 37 oC. Moreover, cells incubated at 37 oC showed increased chances of forming MPECs. In this study, we show that temperature affects CD8+ T cell differentiation into effector phenotype and their cytotoxicity. Further research can provide insight into advanced therapies in cancer treatment and better vaccine efficacy.