Exosomes are small extracellular vesicles secreted by all cell types, playing a key role in intercellular communication by transferring functional proteins, metabolites, and nucleic acids to recipient cells. They are involved in various physiological and pathological processes, including immune responses, tissue repair, and cancer. Exosomes have gained significant interest for clinical applications as diagnostic biomarkers and therapeutic carriers due to their biocompatibility, ability to cross biological barriers, and capacity to deliver genetic cargo. However, challenges remain in precisely targeting cells or organs and minimizing off-target effects. Understanding exosome biology is crucial for their clinical development, including their biogenesis, secretion, transport, uptake, and intracellular signaling. Exosomes are classified into three main types based on size and biogenesis: exosomes (30–200 nm), microvesicles (MVs, 100–1000 nm), and apoptotic bodies (>1000 nm). Exosomes are formed through the endocytic pathway, while MVs are released by budding from the plasma membrane. Exosomes contain proteins, nucleic acids, and lipids, with tetraspanins, ESCRT proteins, and lipid rafts playing key roles in their biogenesis and function. The ESCRT machinery is essential for the formation of intraluminal vesicles (ILVs), which are then secreted as exosomes. Other factors, such as lipid composition and tetraspanins, also influence exosome biogenesis and function. Exosomes are internalized by recipient cells through various mechanisms, including clathrin-mediated endocytosis, lipid raft-mediated endocytosis, caveolin-mediated endocytosis, phagocytosis, and macropinocytosis. Upon internalization, exosomes can either interact with surface receptors to induce signaling or fuse with the plasma membrane to release their contents into the cytosol. Exosome contents can be delivered to the nucleus, endoplasmic reticulum, or lysosomes, where they may be degraded or recycled. Exosomes can also escape lysosomal degradation by bypassing the lysosome through specialized compartments. Exosome research faces challenges in defining and characterizing exosomes, as well as in distinguishing them from other extracellular vesicles. Contamination, heterogeneity, and variability in isolation and characterization methods can lead to inaccurate results. Standardization of exosome research is essential for reliable clinical applications. Understanding exosome biology, including their biogenesis, uptake, and signaling, is key to their successful translation into clinical therapies.Exosomes are small extracellular vesicles secreted by all cell types, playing a key role in intercellular communication by transferring functional proteins, metabolites, and nucleic acids to recipient cells. They are involved in various physiological and pathological processes, including immune responses, tissue repair, and cancer. Exosomes have gained significant interest for clinical applications as diagnostic biomarkers and therapeutic carriers due to their biocompatibility, ability to cross biological barriers, and capacity to deliver genetic cargo. However, challenges remain in precisely targeting cells or organs and minimizing off-target effects. Understanding exosome biology is crucial for their clinical development, including their biogenesis, secretion, transport, uptake, and intracellular signaling. Exosomes are classified into three main types based on size and biogenesis: exosomes (30–200 nm), microvesicles (MVs, 100–1000 nm), and apoptotic bodies (>1000 nm). Exosomes are formed through the endocytic pathway, while MVs are released by budding from the plasma membrane. Exosomes contain proteins, nucleic acids, and lipids, with tetraspanins, ESCRT proteins, and lipid rafts playing key roles in their biogenesis and function. The ESCRT machinery is essential for the formation of intraluminal vesicles (ILVs), which are then secreted as exosomes. Other factors, such as lipid composition and tetraspanins, also influence exosome biogenesis and function. Exosomes are internalized by recipient cells through various mechanisms, including clathrin-mediated endocytosis, lipid raft-mediated endocytosis, caveolin-mediated endocytosis, phagocytosis, and macropinocytosis. Upon internalization, exosomes can either interact with surface receptors to induce signaling or fuse with the plasma membrane to release their contents into the cytosol. Exosome contents can be delivered to the nucleus, endoplasmic reticulum, or lysosomes, where they may be degraded or recycled. Exosomes can also escape lysosomal degradation by bypassing the lysosome through specialized compartments. Exosome research faces challenges in defining and characterizing exosomes, as well as in distinguishing them from other extracellular vesicles. Contamination, heterogeneity, and variability in isolation and characterization methods can lead to inaccurate results. Standardization of exosome research is essential for reliable clinical applications. Understanding exosome biology, including their biogenesis, uptake, and signaling, is key to their successful translation into clinical therapies.