Integrins are crucial for organizing the actin cytoskeleton and signaling from the extracellular matrix. This study reveals a hierarchy of transmembrane responses triggered by integrin aggregation. Integrin aggregation leads to the accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, only tensin among eight cytoskeletal molecules coaccumulates. Integrin aggregation alone activates the JNK pathway, with different kinetics compared to ERK. Tyrosine kinase inhibitors like herbimycin A and genistein block the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. However, pp125FAK continues to coaggregate with α5β1 integrins even in the presence of these inhibitors. Ligand occupancy plus integrin aggregation triggers transmembrane accumulation of three cytoskeletal molecules: talin, α-actinin, and vinculin. These molecules accumulate even in the presence of cytochalasin D. However, four conditions—integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity—are necessary for the accumulation of other cytoskeletal molecules like F-actin and paxillin. Integrins mediate a transmembrane hierarchy of molecular responses. Studies in various cell types, particularly fibroblasts and platelets, have shown that integrin interactions with extracellular ligands lead to intracellular responses. In platelets, ligand occupancy and limited aggregation of αIIbβ3 integrins trigger the first step of intracellular response, while subsequent aggregation due to immobilized ligand or platelet-to-platelet aggregation induces further steps. Fibroblast systems have shown that integrin ligation can trigger activation of certain MAP kinases, specifically ERK members. Other studies have linked ERK activation to growth factor response pathways. SAPK/JNK is not activated by EGF receptors but becomes activated after treatment with inflammatory cytokines or stresses like UV irradiation. Cytoskeletal responses to integrin ligation can be analyzed using beads coated with molecules that interact with integrins. Fibronectin-coated beads induce the local accumulation of various cytoskeletal molecules. The initial integrin response in fibroblasts can be dissected into three parts using soluble ligands vs immobilized multivalent ligands and antibodies that induce integrin aggregation. Ligand occupancy alone, receptor aggregation alone, or a combination of occupancy and aggregation have distinct effects on integrin cytoskeletal function. Ligand occupancy with soluble, monovalent ligand triggers fibroblast integrin localization to preexisting focal contacts but does not trigger FAK phosphorylation. Direct integrin aggregation without ligand occupancy triggers FAK phosphorylation and the local accumulation of FAK and tensin. A combination of integrin aggregation and occupancyIntegrins are crucial for organizing the actin cytoskeleton and signaling from the extracellular matrix. This study reveals a hierarchy of transmembrane responses triggered by integrin aggregation. Integrin aggregation leads to the accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, only tensin among eight cytoskeletal molecules coaccumulates. Integrin aggregation alone activates the JNK pathway, with different kinetics compared to ERK. Tyrosine kinase inhibitors like herbimycin A and genistein block the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. However, pp125FAK continues to coaggregate with α5β1 integrins even in the presence of these inhibitors. Ligand occupancy plus integrin aggregation triggers transmembrane accumulation of three cytoskeletal molecules: talin, α-actinin, and vinculin. These molecules accumulate even in the presence of cytochalasin D. However, four conditions—integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity—are necessary for the accumulation of other cytoskeletal molecules like F-actin and paxillin. Integrins mediate a transmembrane hierarchy of molecular responses. Studies in various cell types, particularly fibroblasts and platelets, have shown that integrin interactions with extracellular ligands lead to intracellular responses. In platelets, ligand occupancy and limited aggregation of αIIbβ3 integrins trigger the first step of intracellular response, while subsequent aggregation due to immobilized ligand or platelet-to-platelet aggregation induces further steps. Fibroblast systems have shown that integrin ligation can trigger activation of certain MAP kinases, specifically ERK members. Other studies have linked ERK activation to growth factor response pathways. SAPK/JNK is not activated by EGF receptors but becomes activated after treatment with inflammatory cytokines or stresses like UV irradiation. Cytoskeletal responses to integrin ligation can be analyzed using beads coated with molecules that interact with integrins. Fibronectin-coated beads induce the local accumulation of various cytoskeletal molecules. The initial integrin response in fibroblasts can be dissected into three parts using soluble ligands vs immobilized multivalent ligands and antibodies that induce integrin aggregation. Ligand occupancy alone, receptor aggregation alone, or a combination of occupancy and aggregation have distinct effects on integrin cytoskeletal function. Ligand occupancy with soluble, monovalent ligand triggers fibroblast integrin localization to preexisting focal contacts but does not trigger FAK phosphorylation. Direct integrin aggregation without ligand occupancy triggers FAK phosphorylation and the local accumulation of FAK and tensin. A combination of integrin aggregation and occupancy