Exosomes are small extracellular vesicles secreted by all cell types and play a critical role in intercellular communication by transferring functional proteins, metabolites, and nucleic acids to recipient cells. They are now being explored for clinical applications as diagnostic biomarkers and therapeutic carriers due to their biocompatibility, ability to cross biological barriers, and capacity to deliver therapeutic cargoes. However, challenges remain in precisely targeting cells and organs while minimizing off-target effects, and in ensuring the purity and functionality of exosomes. Understanding exosome biology is essential for their successful clinical development. Exosomes are generated through the endocytic pathway, involving the formation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), which are then secreted via exocytosis. The biogenesis of exosomes is a complex process involving the ESCRT machinery, which sorts and packages cargo into ILVs. Other factors, including tetraspanins, lipids, and Rab GTPases, also play important roles in exosome formation and secretion. Exosomes are characterized by their membrane-bound proteins, such as tetraspanins (CD9, CD63, CD81), and lipids, including cholesterol, ceramides, and sphingomyelin. They also contain nucleic acids such as mRNA, miRNA, and DNA. Exosomes can be internalized by recipient cells through various mechanisms, including clathrin-mediated endocytosis, lipid raft-mediated endocytosis, caveolin-mediated endocytosis, phagocytosis, and macropinocytosis. Once internalized, exosomes can either fuse with the plasma membrane to release their contents into the cytosol or interact with surface receptors to induce downstream signaling. The intracellular fate of exosomes follows the typical endosomal pathway, leading to their eventual degradation in lysosomes or their release back into the extracellular space. Exosome research is still in its early stages, with significant controversies regarding their definition, characterization, and biogenesis. The heterogeneity of exosomes, influenced by their size, composition, function, and cellular origin, adds complexity to their study. Standardization of exosome isolation, characterization, and reporting is crucial for advancing the field. Despite these challenges, exosomes hold great potential for clinical applications, including diagnostics, therapy, and targeted drug delivery. Understanding the mechanisms of exosome biology is key to overcoming the current limitations and realizing their full therapeutic potential.Exosomes are small extracellular vesicles secreted by all cell types and play a critical role in intercellular communication by transferring functional proteins, metabolites, and nucleic acids to recipient cells. They are now being explored for clinical applications as diagnostic biomarkers and therapeutic carriers due to their biocompatibility, ability to cross biological barriers, and capacity to deliver therapeutic cargoes. However, challenges remain in precisely targeting cells and organs while minimizing off-target effects, and in ensuring the purity and functionality of exosomes. Understanding exosome biology is essential for their successful clinical development. Exosomes are generated through the endocytic pathway, involving the formation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), which are then secreted via exocytosis. The biogenesis of exosomes is a complex process involving the ESCRT machinery, which sorts and packages cargo into ILVs. Other factors, including tetraspanins, lipids, and Rab GTPases, also play important roles in exosome formation and secretion. Exosomes are characterized by their membrane-bound proteins, such as tetraspanins (CD9, CD63, CD81), and lipids, including cholesterol, ceramides, and sphingomyelin. They also contain nucleic acids such as mRNA, miRNA, and DNA. Exosomes can be internalized by recipient cells through various mechanisms, including clathrin-mediated endocytosis, lipid raft-mediated endocytosis, caveolin-mediated endocytosis, phagocytosis, and macropinocytosis. Once internalized, exosomes can either fuse with the plasma membrane to release their contents into the cytosol or interact with surface receptors to induce downstream signaling. The intracellular fate of exosomes follows the typical endosomal pathway, leading to their eventual degradation in lysosomes or their release back into the extracellular space. Exosome research is still in its early stages, with significant controversies regarding their definition, characterization, and biogenesis. The heterogeneity of exosomes, influenced by their size, composition, function, and cellular origin, adds complexity to their study. Standardization of exosome isolation, characterization, and reporting is crucial for advancing the field. Despite these challenges, exosomes hold great potential for clinical applications, including diagnostics, therapy, and targeted drug delivery. Understanding the mechanisms of exosome biology is key to overcoming the current limitations and realizing their full therapeutic potential.