Droplet interface bilayers (DIBs) have recently started to be used as human–mimetic artificial cell membranes. DIBs are bilayer sections created at the interface of two aqueous droplets, such that one droplet can be used as a donor compartment and the other as an acceptor compartment for the quantification of molecular transport across the artificial cell membrane. However, synthetic phospholipids are overwhelmingly used to create DIBs instead of naturally derived phospholipids, even though the diverse distribution of phospholipids in the latter is more biomimetic. We present the first systematic study of the role of temperature in DIB formation, which shows that the temperature at which DIBs are formed is a key parameter for the formation of DIBs using naturally derived phospholipids in a microfluidic platform. The phospholipids that are most abundant in mammalian cell membranes (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)) only form DIBs when the temperature is above the phase transition temperature (T_m). Similarly, DIB formation usually only occurs above the highest T_m of a single phospholipid in a bespoke formulation. In addition, we show a new phenomenon wherein the DIB “melts” without disintegrating for bilayers formed predominantly of phospholipids that occupy cylindrical spaces. We also demonstrate differences in DIB formation rates as well as permeability of these biomimetic membranes. Given the difficulties associated with making DIBs using naturally derived phospholipids, we anticipate this work will illuminate the role of phospholipid phase transition in mono- and bilayer formation and lay the foundation for DIBs to be used as human–mimetic artificial cell membranes.