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Abstract Understanding mesenchymal stromal cells (MSCs) growth mechanisms in response to surface chemistries is essential to optimize culture methods for high‐quality and robust cell yields in cell manufacturing applications. Heparin (HEP) and collagen 1 (COL) substrates have been reported to enhance cell adhesion, growth, viability, and secretory potential in MSCs. However, the biomolecular mechanisms underlying the benefits of combined HEP/COL substrates are unknown. This work used HEP/COL bilayered surfaces to investigate the role of integrin‐HEP interactions in the advantages of MSC culture. The layer‐by‐layer approach (LbL) was used to create HEP/COL bilayers, which were made up of stacks of 8 and 9 layers that combined HEP and COL in an alternate arrangement. Surface spectroscopic investigations and laser scanning microscopy evaluations verified the biochemical fingerprint of each component and a total stacked bilayer thickness of roughly 150 nm. Cell growth and apoptosis in response to IC₅₀and IC₇₅levels of BTT‐3033 and Cilengitide, α2β1 and αvβ3 integrin inhibitors respectively, were evaluated on HEP/COL coated surfaces using two bone marrow‐derived MSC donors. While integrin activity did not affect cell growth rates, it significantly affected cell adhesion and apoptosis on HEP/COL surfaces. HEP‐ending HEP/COL surfaces significantly increased FAK‐ERK½ phosphorylation and endogenous cell COL deposition compared to COL, COL‐ending HEP/COL and uncoated surfaces. BTT‐3033 but not Cilengitide treatment markedly affected FAK‐ERK½ activity levels on HEP‐ending HEP/COL surfaces supporting a major role for α2β1 activity. BTT‐3033 treatment on HEP‐ending bilayers reduced MSC‐mediated macrophage inhibitory activity and altered the cytokine profile of co‐cultures. Overall, this study supports a novel role for HEP in regulating the survival and potency of MSCs via enhancing the α2β1‐FAK‐ERK½ signaling mechanism.