Nano-sized membrane vesicles are secreted by many cell types. These vesicles can serve as carriers of cellular information. DC-derived vesicles can be targeted to other immune cells and modify their function. Accurate analysis of quantitative and qualitative changes in EV production by DC upon different activation stimuli is needed to further reveal the immune regulatory properties of DC-derived EVs. However, methods for reliable quantification of individual EVs and for analysis of the heterogeneity of EV populations are limited. With our recently developed high-resolution flow cytometry-based method, we can perform a high-throughput, multiparameter, and quantitative analysis of individual EVs. With the use of this novel technique, we show that despite previous assumptions, stimulation with bacterial LPS increases EV release by DC. Furthermore, we demonstrate heterogeneity in DC-derived EVs regarding their buoyant density and MHC class II content. Finally, we show that cognate interaction between LPS-stimulated DC and CD4(+) T cells affects both the quantity and quality of LPS DC-derived EVs present in the culture supernatant. These data indicate that flow cytometry-based analysis of individual EVs is a valuable, novel tool to study the dynamics of EV secretion and composition, offering great opportunities to unveil the function of immune cell-derived EVs.