Immune cells release microRNA-containing exosomes that can be taken up by recipient cells. Exosomes can thus act as mediators of cell–cell communication through direct exchange of genetic material between cells. Exosome-mediated transfer of miRNAs between T cells and antigen-presenting cells (APCs) can take place over long distances. Our work has shown that this transfer is enhanced by the formation of a functional immune synapse. Here we give a detailed description of the isolation of exosomes produced by immune cells by ultracentrifugation, their quantification by flow cytometry, and the analysis of miRNA and protein exchange between T cells and APCs, both at a distance and after the formation of an immune synapse.