Extracellular vesicles (EVs), including exosomes, are increasingly recognized as potent mediators of intercellular communication due to their ability to transport a diverse array of bioactive molecules. Exosomes, a subset of EVs, are small membrane-bound vesicles that originate from the endocytic pathway and play crucial roles in physiological and pathological processes. They contain a variety of cargo molecules, including proteins, lipids, nucleic acids, and signaling molecules, which determine their targeting specificity and functional roles upon reaching recipient cells. Despite growing understanding of exosomes' significance in biological processes, the molecular mechanisms governing their cargo selection and packaging remain unclear. This review summarizes current insights into the molecular mechanisms regulating exosome cargo sorting, their biological functions, and potential clinical applications, with a focus on their relevance in cancer and other diseases. Exosome biogenesis is a complex, highly regulated process involving several sequential stages, from the formation of early endosomes to the release of mature exosomes. The selective sorting of cargo molecules into nascent exosome vesicles is a critical determinant of exosome functionality, diversity, and specificity. The review explores the various classes of molecules that constitute exosome cargo, the regulatory factors and machinery that govern cargo selection and packaging, and the implications of cargo sorting in disease contexts. Understanding how exosomes manage cargo sorting is crucial for disease management and a foundational step in advancing exosome-based therapies. Exosomes are rich in various forms of nucleic acids, including mRNAs, miRNAs, rRNAs, lncRNAs, tRNAs, snRNAs, snoRNAs, and piRNAs. They also contain double-stranded or single-stranded DNA and mitochondrial DNA. The primary membrane-bound and cytosolic proteins incorporated in exosomes include those involved in membrane transport or fusion, proteins associated with exosome biogenesis, heat shock proteins, integrins, members of the tetraspanin family, and cytoskeletal proteins. Numerous antigen and receptor proteins are expressed on the surface of exosomes, which interact with receptors on recipient cells to initiate intracellular signaling pathways. Exosome biogenesis involves the formation of intraluminal vesicles (ILVs) within endocytic compartments known as multivesicular bodies (MVBs). These ILVs are enriched with specific cargoes and interact with trafficking effectors on endosomal and plasma membrane patches. These interactions lead to membrane bending and scission processes that give rise to exosomes. The ESCRT pathway is one of the first pathways associated with exosome biogenesis and involves the coordinated action of all four ESCRT complexes. The ESCRT pathway is crucial for the formation of ILVs and the subsequent release of exosomes. However, there are also ESCRT-independent mechanisms that govern exosome generation, such as the role of ceramide in exosome biogenesis. Exosomes also serve as vehicles for DNA and mtDNA transport. Various DNA species, including genomic dsExtracellular vesicles (EVs), including exosomes, are increasingly recognized as potent mediators of intercellular communication due to their ability to transport a diverse array of bioactive molecules. Exosomes, a subset of EVs, are small membrane-bound vesicles that originate from the endocytic pathway and play crucial roles in physiological and pathological processes. They contain a variety of cargo molecules, including proteins, lipids, nucleic acids, and signaling molecules, which determine their targeting specificity and functional roles upon reaching recipient cells. Despite growing understanding of exosomes' significance in biological processes, the molecular mechanisms governing their cargo selection and packaging remain unclear. This review summarizes current insights into the molecular mechanisms regulating exosome cargo sorting, their biological functions, and potential clinical applications, with a focus on their relevance in cancer and other diseases. Exosome biogenesis is a complex, highly regulated process involving several sequential stages, from the formation of early endosomes to the release of mature exosomes. The selective sorting of cargo molecules into nascent exosome vesicles is a critical determinant of exosome functionality, diversity, and specificity. The review explores the various classes of molecules that constitute exosome cargo, the regulatory factors and machinery that govern cargo selection and packaging, and the implications of cargo sorting in disease contexts. Understanding how exosomes manage cargo sorting is crucial for disease management and a foundational step in advancing exosome-based therapies. Exosomes are rich in various forms of nucleic acids, including mRNAs, miRNAs, rRNAs, lncRNAs, tRNAs, snRNAs, snoRNAs, and piRNAs. They also contain double-stranded or single-stranded DNA and mitochondrial DNA. The primary membrane-bound and cytosolic proteins incorporated in exosomes include those involved in membrane transport or fusion, proteins associated with exosome biogenesis, heat shock proteins, integrins, members of the tetraspanin family, and cytoskeletal proteins. Numerous antigen and receptor proteins are expressed on the surface of exosomes, which interact with receptors on recipient cells to initiate intracellular signaling pathways. Exosome biogenesis involves the formation of intraluminal vesicles (ILVs) within endocytic compartments known as multivesicular bodies (MVBs). These ILVs are enriched with specific cargoes and interact with trafficking effectors on endosomal and plasma membrane patches. These interactions lead to membrane bending and scission processes that give rise to exosomes. The ESCRT pathway is one of the first pathways associated with exosome biogenesis and involves the coordinated action of all four ESCRT complexes. The ESCRT pathway is crucial for the formation of ILVs and the subsequent release of exosomes. However, there are also ESCRT-independent mechanisms that govern exosome generation, such as the role of ceramide in exosome biogenesis. Exosomes also serve as vehicles for DNA and mtDNA transport. Various DNA species, including genomic ds