The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the β1 integrin family regulate myoblast fusion. β1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of β1-deficient myoblasts, suggesting that β1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, β1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of β1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.