Many cell types release nano-sized vesicles, which can be found in body fluids as well as in cell culture-conditioned medium. These extracellular vesicles (EV) have been identified as vehicles for intercellular communication and are thought to be involved in many (patho)physiological processes. They have also gained interest due to their potential clinical application as biomarkers for disease and as therapeutic agents. However, the exact mechanisms through which EV exert their functions are largely unknown. EV carry a selective set of proteins, lipids and RNA. The molecular composition of EV and the dynamics of their release depends on the status of the producing cell. The released EV are heterogeneous in size and composition. Knowledge on how the molecular composition and number of released EV is regulated upon different triggers is needed to gain more insight in the physiological role of EV in intercellular communication. In her thesis, Els van der Vlist focused on EV that are released by dendritic cells (DC) and T cells, in order to unravel the role of these EV in immune cell communication. Her thesis describes a newly developed high-resolution flow cytometric method enabling simultaneous quantitative and qualitative analysis of individual EV. With this method, large numbers of nano-sized EV can be analyzed, which is needed to reveal phenotypic heterogeneity within EV populations. The method was used to analyze the quantity and quality of dendritic cell (DC) and T cell-derived EV subsets that were released upon different activation triggers. Using this approach, it was demonstrated that DC release more EV, with altered molecular compositions, in response to activation with bacterial lipopolysaccharide (LPS). Furthermore, it was observed that when DC were cultured together with CD4+ T cells, these T cells selectively recruited a subset of DC-derived EV. CD4+ T cells also increased their release of EV when activated via their T cell receptor, and additional co-stimulatory signals potentiated this process further. Interestingly, using the flow cytometric analysis method it was found that the population of EV released by CD4+ T cells was composed of different vesicle subsets. The release of these subsets was differentially regulated in response to the level of T cell activation. Finally, the function of vesicles released during cognate DC-T cell interactions was investigated in an in vitro model system. EV released during strong stimulatory DC-T cell interactions are targeted to neighboring DC and T cells and can influence the phenotype of these cells. Thus, EV that are released during strong stimulatory DC-T cell interactions might be involved in the modulation of neighboring DC-T cell interactions. The novel individual EV-based flow cytometric method has increased our insights in the regulated release of different vesicle subsets by DC and T cells and will greatly facilitate further research to unravel the function of EV in intercellular communication. For clinical settings this method holds great promise for the definition and analysis of EV-based biomarkers and therapeutics.