ABCB4 flops phosphatidylcholine into the bile canaliculus to protect the biliary tree from the detergent activity of bile salts. Homozygous-null ABCB4 mutations cause the childhood liver disease, progressive familial intrahepatic cholestasis, but cause and effect is less clear, with many missense mutations linked to less severe cholestatic diseases. ABCB4S320F, in particular, is described in 13 patients, including in heterozygosity with ABCB4A286V, ABCB4A953D, and null mutants, whose symptoms cover the spectrum of cholestatic disease. We sought to define the impact of these mutations on the floppase, explain the link with multiple conditions at the molecular level, and investigate the potential for reversal. ABCB4S320F, ABCB4A286V, and ABCB4A953D expression was engineered in naïve cultured cells. Floppase expression, localization, and activity were measured by western blot, confocal microscopy, and lipid transport assays, respectively. ABCB4S320F was fully active for floppase activity but expression at the plasma membrane was reduced to 50%. ABCB4A286V expressed and trafficked efficiently but could not flop lipid, and ABCB4A953D expressed poorly and was impaired in floppase activity. Proteasome inhibition stabilized nascent ABCB4S320F and ABCB4A953D but did not improve plasma membrane localization. Cyclosporin-A improved plasma membrane localization of both ABCB4S320F and ABCB4A953D, but inhibited floppase activity. Conclusion: The level of ABCB4 functionality correlates with, and is the primary determinant of, cholestatic disease severity in these patients. ABCB4S320F homozygosity, with half the normal level of ABCB4, is the tipping point between more benign and potentially fatal cholestasis and makes these patients more acutely sensitive to environmental effects. Cyclosporin-A increased expression of ABCB4S320F and ABCB4A953D, suggesting that chemical chaperones could be exploited for therapeutic benefit to usher in a new era of personalized medicine for patients with ABCB4-dependent cholestatic disease. (Hepatology 2014;59:1921–1931)