Kindlin family members are essential for supporting integrin activation by functionally cooperating with the talin head domain. Both the talin head and kindlin are FERM domain-containing proteins that can simultaneously interact with the integrin beta cytoplasmic tails. While the talin head is well studied, the molecular basis of kindlin's interaction with integrin during integrin activation is still poorly understood. Here we defined the subdomain boundaries in kindlin-2 and evaluated their contribution to integrin activation and recognition. We found that each subdomain in kindlin-2 was required for co-activating the integrin alpha IIb beta 3 together with the talin head (inside-out signaling) and for enhancing integrin alpha IIb beta 3-mediated cell spreading (outside-in signaling). To evaluate the involvement of the kindlin-2 subdomains in integrin interaction, we developed a FACS-based binding assay and found that an intact FERM domain in kindlin-2 was required for the interaction. Taking all together, these findings suggest that the integrity of kindlin-2 subdomains is a prerequisite for supporting integrin recognition and for subsequent integrin activation. (C) 2013 Elsevier Inc. All rights reserved.