Protein transport in the secretory and endocytic pathways is a multistep process involving the generation of transport carriers loaded with defined sets of cargo, the shipment of the cargo-loaded transport carriers between compartments, and the specific fusion of these transport carriers with a target membrane. The regulation of these membrane-mediated processes involves a complex array of protein and lipid interactions. As the machinery and regulatory processes of membrane trafficking have been defined, it is increasingly apparent that membrane transport is intimately connected with a number of other cellular processes, such as quality control in the endoplasmic reticulum (ER), cytoskeletal dynamics, receptor signaling, and mitosis. The fidelity of membrane trafficking relies on the correct assembly of components on organelles. Recruitment of peripheral proteins plays a critical role in defining organelle identity and the establishment of membrane subdomains, essential for the regulation of vesicle transport. The molecular mechanisms for the biogenesis of membrane subdomains are also central to understanding how cargo is sorted and segregated and how different populations of transport carriers are generated. In this review we will focus on the emerging themes of organelle identity, membrane subdomains, regulation of Golgi trafficking, and advances in dissecting pathways in physiological systems.