The generation of skeletal muscle fibers is achieved by cell-cell fusion. Mononucleated myoblasts fuse to form multinucleated myotubes in the primary stages of muscle development. During myoblast fusion the plasma membrane undergoes lipid topology changes. However little is known about the lipid transporters involved in regulating those changes. It is important to advance such knowledge for the treatment of devastating diseases such as muscular dystrophy. By using the mouse myoblast C2C12 cell line as cellular model system, the first stage of this project involved a detailed kinetic study of the transbilayer movement of NBD-labeled phospholipids in the plasma membrane of proliferating and differentiating myoblasts. Proliferating C2C12 cells exhibited a fast, selective uptake of aminophospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane, which was downregulated during cell fusion. Expression analysis revealed the presence of five P4-ATPases (ATP8B2, ATP10A, ATP10D, ATP11A, and ATP11C) and one CDC50 protein (CDC50A) that could be involved in the regulation of the phospholipid dynamics in the plasma membrane of myoblasts. Among these, CDC50A, ATP11A, and ATP8B2 were further investigated. To test their role in myoblast fusion, these genes were knocked out by the CRISPR/Cas9 approach. Three clones of each knockout cell line were isolated and further characterized. CDC50A-deficient cells completely lost the ability to flip aminophospholipids across the plasma membrane and to form myotubes. ATP11A-deficient cells displayed a decrease of phosphatidylethanolamine- and phosphatidylserine-flipping activity. Although their fusion was delayed, ATP11A-deficient cells still formed myotubes similar to wild-type cells. ATP8B2-deficient cells displayed a similar phospholipid uptake to wild-type cells, but presented changes in their myotube shape. These findings demonstrate that lipid transporters of the P4-ATPase-CDC50 family play a crucial role in myoblasts fusion. The results advance the understanding of molecular mechanisms underlying muscle cell development and provide insights into how the failure of these mechanisms may give rise to pathologies.