Gradient surfaces are highly effective tools to screen and optimize cell– surface interactions. Here, the response of embryonic stem (ES) cell colonies to plasma polymer gradient surfaces is investigated. Surface chemistry ranged from pure allylamine (AA) plasma polymer on one end of the gradient to pure octadiene (OD) plasma polymer on the other end. Optimal surface chemistry conditions for retention of pluripotency were identified. Expression of the stem cell markers alkaline phosphatase (AP) and Oct4 varied with the position of the ES cell colonies across the OD-AA plasma polymer gradient. Both markers were more strongly retained on the OD plasma polymer rich regions of the gradients. The observed variation of expression across the plasma polymer gradient increased with duration of stem cell culture. While maximum cell adhesion to the gradient substrate occurred at a nitrogen- to-carbon (N/C ratio) of approximately 0.1, Oct4 and AP expression was best retained at an N/C ratio < 0.04. Stem cell marker expression correlated with colony size and morphology: more compact, multilayered colonies with prominent F-actin staining arose as the N/C ratio decreased. Disruption of actin polymerization using Y-27632 ROCK inhibitor resulted in a collapse of the multilayer colony structure into monolayers with limited cell–cell contact. A corresponding decrease in expression of AP and Oct4 was observed. Oct4 expression along with 3D colony morphology was partially rescued on the OD plasma polymer rich regions of the gradient.