We report the development of a photo-reversible cell culture substrate. We demonstrate the capacity to modify the adhesivity of the substrate using light, altering its capacity to support cell growth. Polyelectrolyte multilayers (PEMs) were used to produce tunable substrates of different thickness and matrix stiffness, which have different intrinsic capacities to support cell adhesion and survival. Surfaces were top-coated with a poly(acrylic acid)-poly(allylamine hydrochloride) polyelectrolyte bilayer functionalized with a small fraction (<1%) of an azobenzene-based photoswitchable sidegroup, which included the cell-adhesive three-amino-acid peptide RGD. Irradiation with light-induced geometric switching of the azo bond, resulting in changes to RGD exposure and consequently to cell adhesion and survival, was investigated on a variety of surfaces of different thickness and stiffness. Substrate stiffness, as modified by the thickness, had a significant influence on the adhesion of NIH 3T3 cells, consistent with previous studies. However, by disrupting the isomerization state of the azobenzene-linked :RGD and exposing it to the surface, cell adhesion and survival could be enhanced up to 40% when the positioning of the RGD peptide was manipulated on the softest substrates. These findings identify permissive, yet less-than-optimal, cell culture substrate conditions that can be substantially enhanced using noninvasive modification of the substrate triggered by light. Indeed, where cell adhesion was tuned to be suboptimal under baseline conditions, the light-induced triggers displayed the most enhanced effect, and identification of this 'Goldilocks zone' was key to enabling light triggering.