Lipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fungi. The encapsulated fungal pathogen Cryptococcus neoformans contains four P4-ATPases, among which Apt2-4p are poorly characterized. Using heterologous expression in the flippase-deficient S. cerevisiae strain dnf1Δdnf2Δdrs2Δ, we tested their lipid flippase activity in comparison to Apt1p using complementation tests and fluorescent lipid uptake assays. Apt2p and Apt3p required the co-expression of the C. neoformans Cdc50 protein for activity. Apt2p/Cdc50p displayed a narrow substrate specificity, limited to phosphatidylethanolamine and –choline. Despite its inability to transport fluorescent lipids, the Apt3p/Cdc50p complex still rescued the cold-sensitive phenotype of dnf1Δdnf2Δdrs2Δ, suggesting a functional role for the flippase in the secretory pathway. Apt4p, the closest homolog to Saccharomyces Neo1p, which does not require a Cdc50 protein, was unable to complement several flippase-deficient mutant phenotypes, neither in the presence nor absence of a β-subunit. These results identify C. neoformans Cdc50 as an essential subunit for Apt1-3p and provide a first insight into the molecular mechanisms underlying their physiological functions.