Cells interact with ECM via membrane proteins. Especially integrins, which are known as mechanoreceptor proteins, transmit mechanical cues from environment to intracellular signaling pathways via cytoskeleton. There are multiple types of integrin dimers present on the skin cell membrane. Depending on their types, each integrin is associated with different extracellular matrix (ECM) proteins such as fibronectin (FN), collagen, laminin, and various other types of cell adhesion molecules (CAM). In this study, we tested three different types of ECM proteins, FN, collagen and gelatin. In order to apply different mechanical cues, we used polystyrene, PDMS and different percentage of polyacrylamide gel to change substrate stiffness. Results show that actin stress fiber and paxillin expression level depends on substrate stiffness and ECM proteins, especially it was significantly observed when cells are on FN coating condition. Furthermore, the mRNA expression levels of epidermal differentiation proteins also depend on these two factors. Our results suggested that skin cells’ response to the substrate stiffness and differentiation depend on the ECM coating material and thus the linker integrin types.