If the binding strength of adhesive cues to the extracellular matrix (ECM) and the mechanisms involved in cell adhesion are synergistically correlated via a “mechanical” feedback, engineering of cue presentation at the ECM by designer macromolecules can enable control over cell–matrix interaction. Here, polymer brushes supporting fibronectin (FN) and presenting different grafted-chain length to modulate cell interaction at ECM cell-binding sites are exploited. Application of friction force microscopy allows us to estimate the lateral deformability and friction of oligoethylene glycol-containing brushes. These parameters are demonstrated to regulate the adhesion of human mesenchymal stem cells (hMSCs), which adopt their morphology and form focal adhesions (FAs) responding to FN brush-tether characteristics. Across a brush-thickness gradient presenting uniform FN exposure, thin brushes stimulate cell spreading and the development of FAs. Conversely, thick and more laterally deformable polymer grafts induce a decrease in cell spreading and FA formation. A correlation between frictional forces experienced at the (macro)molecular scale and the behavior of stem cells has been found. This interaction can be clarified by exploring novel aspects of FFM, which demonstrates a powerful tool to dynamically probe the ECM environment and indirectly suggest a way to structure ECM in order to trigger specific cell responses.