The Arabidopsis ATP-binding cassette B19 (ABCB19, P-glycoprotein19) transporter functions coordinately with ABCB1 and PIN1 to motivate long-distance transport of the phytohormone auxin from the shoot to root apex. ABCB19 exhibits a predominantly apolar plasma membrane (PM) localization and stabilizes PIN1 when the two proteins co-occur. Biochemical evidence associates ABCB19 and PIN1 with sterol- and sphingolipid-enriched PM fractions. Mutants deficient in structural sterols and sphingolipids exhibit similarity to abcb19 mutants. Sphingolipid-defective tsc10a mutants and, to a lesser extent, sterol-deficient cvp1 mutants phenocopy abcb19 mutants. Live imaging studies show that sterols function in trafficking of ABCB19 from the trans-Golgi network to the PM. Pharmacological or genetic sphingolipid depletion has an even greater impact on ABCB19 PM targeting and interferes with ABCB19 trafficking from the Golgi. Our results also show that sphingolipids function in trafficking associated with compartments marked by the VTI12 syntaxin, and that ABCB19 mediates PIN1 stability in sphingolipid-containing membranes. The TWD1/FKBP42 co-chaperone immunophilin is required for exit of ABCB19 from the ER, but ABCB19 interactions with sterols, sphingolipids and PIN1 are spatially distinct from FKBP42 activity at the ER. The accessibility of this system to direct live imaging and biochemical analysis makes it ideal for the modeling and analysis of sterol and sphingolipid regulation of ABCB/P-glycoprotein transporters.