Updated project metadata. Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signaling factors in EVs contributes to organ development and tissue differentiation. Here, we present an in vivo model to study EV secretion using the fat body and the hemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualization of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterization of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. We furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV trafficking and secretion.