||Liposomes are small vesicles filled with an aqueous solvent, bounded by a fluid shell that is comprised of a lipid bilayer. The bilayer may contain a single lipid species or a complex mixture that approximates the lipid content of a specific cellular membrane. This research developed a microfluidic and a biological framework for creating and probing the functionality of synthetic vesicles. To study vesicles that either approximated the size of cells or endosomal compartments, a microfluidic device was developed capable of creating nano- or microscale vesicles. This was the first demonstration of a microfluidic strategy able to access both size ranges. This technique enabled the discovery of novel functionality of several ESCRT-III (Endosomal Sorting Complex Required for Transport) proteins while also enabling the first in-depth electron microscopy analysis of liposomes created by a microfluidic platform. To encourage adoption of the microfluidic technique developed in this work, a three-dimensional (3D) printed framework was developed for manufacturing leak-free, transparent microfluidic devices. The methodology developed in this section led to the creation of the smallest ever FDM (Fused Deposition Modeling) 3D printed microfluidic channels (400 μm x 400 μm), demonstrating leak-free operation up to ~5MPa. To increase heat resistance of these devices, an annealing technique was developed for PLA (Poly(lactic Acid)). This was used to demonstrate the first DNA melting analysis on a 3D printed microfluidic device capable of withstanding temperatures up to 100ºC. iv A new method was developed for the 3D printing of 200 μm x 200 μm channels, the smallest to date using an FDM style printer. In conjunction with this method, a technique was developed for direct integration of a polycarbonate membrane into a 3D printed device. The entirely 3D printed protocol was used to demonstrate continuous formation of vesicles. This was the first demonstration of vesicles synthesized using a 3D printed platform. This technique enabled encapsulation of high ionic strength solutions in cell-sized vesicles (~5 μm), created in a low viscosity fluid.