Sensing of extracellular matrix (ECM) physical properties is critical for several processes such as cell differentiation, cell migration in the developmental (haptotaxis) and disease (cancer metastasis). Clustering of matrix-activated integrins is an important step in the formation of matrix adhesions and clustering can occur by lateral association of activated integrin receptors. Using supported bilayers with fluid, lipid-linked RGD ligands, large clusters of activated integrins formed in bound mouse embryo fibroblasts (Yu et al., 2011. PNAS 108:20585). After cells spread on RGD bilayers for 15 minutes, integrin clusters were formed even in the presence of inhibitors of cytoskeletal assembly. We have quantified the cluster size, geometry and receptor distribution at a nanometer level using photoactivated light microscopy. When the temperature was decreased to 250C, the cluster formation was greatly diminished, indicating that the integrin cluster formation may be an entropically driven process. To identify the players crucial for this process, we observed that when Talin1 knockout cells spread, the cluster size and the position of the clusters around the cell was altered. This was rescued by a full length Talin construct, indicating that Talin is required for the early cluster formation. This was also rescued by Talin head domain and not by Talin rod domain, indicating that most likely in the absence of external force, the integrin activation by Talin head was required for cluster to form. This study shows that the integrin cluster formation proceeds in the absence of external traction force in a Talin1 dependent manner. Thus, we suggest that talin head binding is an important factor in integrin clustering.