Integrin receptors play crucial roles in organizing the actin-containing cytoskeleton and signal transduction from the extracellular matrix. This study investigates the molecular hierarchies of transmembrane actions triggered by integrin aggregation. Simple integrin aggregation leads to the localized accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, only tensin accumulates among the tested cytoskeletal molecules. Integrin aggregation alone is sufficient to induce rapid activation of the JNK pathway, with kinetics different from those of ERK. Tyrosine kinase inhibitors herbimycin A and genistein block the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D has similar effects, while other tyrosine kinase inhibitors do not. The kinase pp125FAK continues to coaggregate with α5β1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition does not block the transmembrane accumulation of three cytoskeletal molecules (talin, α-actinin, and vinculin) triggered by ligand occupancy plus integrin aggregation. However, all four conditions—integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity—are necessary for the accumulation of other cytoskeletal molecules, including F-actin and paxillin. Integrins thus mediate a transmembrane hierarchy of molecular responses. The study also explores the effects of inhibitors on integrin-induced signaling and cytoskeletal protein complexes, revealing distinct hierarchies of independence or dependence on tyrosine phosphorylation, actin microfilament integrity, and ligand occupancy.Integrin receptors play crucial roles in organizing the actin-containing cytoskeleton and signal transduction from the extracellular matrix. This study investigates the molecular hierarchies of transmembrane actions triggered by integrin aggregation. Simple integrin aggregation leads to the localized accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, only tensin accumulates among the tested cytoskeletal molecules. Integrin aggregation alone is sufficient to induce rapid activation of the JNK pathway, with kinetics different from those of ERK. Tyrosine kinase inhibitors herbimycin A and genistein block the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D has similar effects, while other tyrosine kinase inhibitors do not. The kinase pp125FAK continues to coaggregate with α5β1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition does not block the transmembrane accumulation of three cytoskeletal molecules (talin, α-actinin, and vinculin) triggered by ligand occupancy plus integrin aggregation. However, all four conditions—integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity—are necessary for the accumulation of other cytoskeletal molecules, including F-actin and paxillin. Integrins thus mediate a transmembrane hierarchy of molecular responses. The study also explores the effects of inhibitors on integrin-induced signaling and cytoskeletal protein complexes, revealing distinct hierarchies of independence or dependence on tyrosine phosphorylation, actin microfilament integrity, and ligand occupancy.