Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane. One mammalian phospholipid flippase at the plasma membrane is ATP11C, a type IV P-type ATPase (P4-ATPase) that forms a heterocomplex with the transmembrane protein CDC50A. However, the structural features in CDC50A that support the function of ATP11C and other P4-ATPases have not been characterized. Here, using error-prone PCR-mediated mutagenesis of human CDC50A cDNA followed by functional screening and deep sequencing, we identified 14 amino acid residues that affect ATP11C's flippase activity. These residues were all located in CDC50A's extracellular domain and were evolutionarily well-conserved. Most of the mutations decreased CDC50A's ability to chaperone ATP11C and other P4-ATPases to their destinations. The CDC50A mutants failed to form a stable complex with ATP11C and could not induce ATP11C's PtdSer-dependent ATPase activity. Notably, one mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity. These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A's ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C's ATP hydrolysis-coupled flippase activity.