Exosomes are small (30-150 nm) extracellular vesicles (EVs) that obtain their membrane through the inward invagination of the endosomes, which are known as multivesicular endosomes (MVEs). Exosomes are secreted to the extracellular milieu when MVEs dock and fuse with the plasma membrane. However, MVEs can also fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane rather than to lysosomes is unclear. My thesis work mainly focused on the molecular mechanisms that control the exocytic trafficking of MVEs and, ultimately, exosome secretion. We found that the conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. Disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes.In collaboration with the lab members, we also found that cells respond to extracellular matrix (ECM) stiffness for exosome secretion. Increased ECM stiffness activates Akt, which directly phosphorylates Rabin8 and promotes its guanine nucleotide exchange activity toward Rab8 for the exocytic trafficking of MVEs and exosome secretion. Together, my work suggests that exocytic trafficking of MVEs and exosome secretion is mediated by phospholipids, Rab GTPases, and the exocyst complex, which is regulated by signaling molecules in response to extracellular cues.