Infections are often accompanied by fever. But what effect does this increase in temperature have on immune cell activation? This study found that incubation of naive CD8+ T cells at 39.5°C prior to antigen-specific activation resulted in enhanced differentiation of these cells into effector CD8+ T cells compared with cells incubated at 33°C or 37°C. Furthermore, enhanced naive CD8+ T cell differentiation was observed in mice following whole-body hyperthermia. But, the rate of T cell proliferation was not affected. However, clustering of GM1+ cholesterol-dependent microdomains on CD8+ T cells was increased at 39.5°C both in vitro and in vivo, as was the rate of T cell–antigen-presenting cell (APC) conjugate formation. So, physiologically relevant increases in temperature enhance the antigen-specific differentiation of effector CD8+ T cells by increasing T cell–APC conjugation.