Lipid bilayer membranes are ubiquitous in biology and electrostatics play a key role in their functionality. The interfacial electrostatics of lipid bilayers involves interplay between the surface potential and charge regulation in the form of ion binding, protonation and lipid mobility. Mobile lipid charge regulation in particular is unique to lipid interfaces and is thought to be an important factor in charged macromolecule-membrane interactions. We used Atomic Force Microscopy (AFM) for the first nanometer scale experimental demonstration of mobile lipid charge regulation occurring in supported lipid bilayer membranes. By combining finite element computer simulations and experimental AFM data, we showed that mobile lipid charge regulation accounts for the short range deviations from the expected electrostatics over anionic lipids. We also accounted for van der Waal interactions and electrolyte ion binding in our calculations and found the mobility of the lipid to be the dominant factor in the short range deviations. Control experiments on silicon nitride surfaces, whose surface charges are immobile, showed that the short range deviation could be accounted for by the formation of a stem layer due to cation binding. Further evidence for tip-induced mobile lipid charge regulation was presented in the form of clear differences in the short range electrostatics of mobile fluid phase lipids when compared to immobile gel phase lipids. Furthermore, our data confirmed the theoretically predicted differences between surfaces containing mobile versus immobile charges.