The mixed leukocyte reaction (MLR) is a crucial in vitro functional assay to evaluate T-cell priming, specifically their activation and clonal expansion when stimulated with antigen presenting cells. The use of monocyte-derived dendritic cells (moDCs) in this assay is particularly noteworthy as they can induce antigen-specific responses using autologous T cells or alloreactive responses using allogenous T cells and stimulating both naïve and/or memory. These outstanding attributes make moDCs a valuable tool for assessing alloreactivity, such as before allogeneic stem cell transplantation (and the potential development of graft-versus-host disease) or when required to analyze the impact of external factors (such as suppressive drugs) on T-cell responses. This chapter presents a comprehensive protocol for isolating peripheral blood human monocytes, differentiating them into moDCs in vitro, and applying them in an MLR assay to evaluate alloreactive memory T-cell responses using flow cytometry.