The release of encapsulated cargo is an important step towards the design of intelligent drug delivery systems or vesicle-based bioreactors. Controlling the integration of pore forming channels into phospholipid vesicle membranes provides a route to initiate content release that could be coupled to gene-expression systems. Here, we present the design of phospholipid vesicles that release cargo upon the temporally controlled assembly of pore-forming channels. Our approach was to design a synthetic vesicle in which membrane composition could be remotely changed to enable the assembly of a pore-forming channel protein, α-hemolysin (αHL), and trigger content release. We first observed that a membrane lacking a sufficient concentration of cholesterol is not compatible with channel assembly and we hypothesized that increasing membrane fluidity, through the addition of oleic acid (OA) micelles could promote channel assembly. Using a dye release assay, we first confirmed that increasing OA content in phospholipid membranes leads to pore assembly. Next, we demonstrated that remote addition of OA micelles to inactive membranes results in fatty acid membrane uptake and channel assembly. We then showed that our system provides a route to control the integration of αHL into vesicle membranes when the channel protein is produced through a cell-free expression system. We remotely added OA micelles to vesicles present in a cell-free expression system where the amount of protein expressed is difficult to control. OA addition led to functional pore assembly and dye release. We ultimately demonstrate a new way to utilize remote changes in membrane composition to control αHL assembly and content release. Our study contributes to the design of vesicle-based technologies and also reveals new insight into the relationship between membrane composition and cholesterol-dependent cytolysin channel assembly.