AbstractAKT-inhibition is a promising approach to improve T cell therapies; however, its effect on CD4⁺ T cells is insufficiently explored. Previously, we and others showed that AKT-inhibition during ex vivo CD8⁺ T cell expansion facilitates the generation of polyfunctional T cells with stem cell memory-like traits. However, most therapeutic T cell products are generated from lymphocytes, containing CD4⁺ T cells that can affect CD8⁺ T cells dependent on the Th-subset. Here, we investigated the effect of AKT-inhibition on CD4⁺ T cells, during separate as well as total T cell expansions. Interestingly, ex vivo AKT-inhibition preserved the early memory phenotype of CD4⁺ T cells based on higher CD62L, CXCR4 and CCR7 expression. However, in the presence of AKT-inhibition, Th-differentiation was skewed toward more Th2-associated at the expense of Th1-associated cells. Importantly, the favorable effect of AKT-inhibition on the functionality of CD8⁺ T cells drastically diminished in the presence of CD4⁺ T cells. Moreover, also the expansion method influenced the effect of AKT-inhibition on CD8⁺ T cells. These findings indicate that the effect of AKT-inhibition on CD8⁺ T cells is dependent on cell composition and expansion strategy, where presence of CD4⁺ T cells as well as polyclonal stimulation impede the favorable effect of AKT-inhibition.