Extracellular vesicles (EVs) are released by all cells, and they regulate intercellular communication in health and disease. A subset of EVs, exosomes, play significant roles in cellular communication and influence a broad range of physiological processes. Exosomes have gained increasing attention in clinical setting due to their ability to transfer cargoes, including signalling mediators, to recipient cells. Exosomes – defined as membrane-bound EVs 30-200 nm in size – originate from multivesicular bodies (MVBs), a specialised endosomal compartment rich in intraluminal vesicles (ILVs). The process of forming ILVs and secreting their contents at the plasma membrane depends on the combined function of a variety of cellular components. Exosomes can trigger signalling in target cells either by direct interaction with extracellular receptors or by fusing with the plasma membrane or internalisation of exosome contents can occur through a variety of pathways. Controversies exist in exosome research, including whether exosomes are truly only derived from endosomal pathways and what determines the heterogeneous content of exosomes within a cell and exosome research is complex, requiring considerations for multifunctional cell signalling pathways and the potential for artefacts due to storage and contamination issues. Better understanding of the intricate pathways involved in exosome biology will help ensure the successful development of exosome-based therapies.