Plasmacytoid dendritic cells (pDCs) are critical in antiviral immune responses and play a significant role in developing autoimmune diseases and transplant rejection. Despite their scarcity in peripheral blood, the accessibility of blood peripheral pDCs makes them a promising source for evaluating T-cell antigen-specific responses in viral infections and autoimmune diseases, as well as alloreactive stimulation in transplantation, including graft-versus-host disease. T-cell priming assays with pDCs have presented a significant challenge due to the low proliferation of T cells. This underscores the critical need for meticulous standardization of suitable conditions for cell purification and coculture in peripheral blood pDC-based mixed leukocyte reactions (MLRs) to ensure productive and quantitative results. This chapter presents a thorough methodology for isolating peripheral blood human pDCs and their application in an MLR assay to evaluate alloreactive T-cell responses using flow cytometry and the adjustments to analyze antigen-specific reactions.