Eukaryotic cell membranes are built from a wide variety of lipid species. These lipids play multiple critical roles, including maintaining membrane permeability properties, signaling, protein recruitment and regulation, and induction of membrane curvature. Phosphoinositides (PIPs) are a class of phospholipids on cytosolic membrane leaflets defined by their glycerol backbone and myo-inositol ring headgroup, which can undergo combinatorial phosphorylation at positions 3, 4 and 5, resulting in the formation of seven different phosphoinositide species. PI4KIIIα is the major enzyme responsible for generating the phosphoinositide PI(4)P at the plasma membrane (PM). This lipid kinase forms two multicomponent complexes, both of which include the palmitoylated membrane anchor, EFR3. Whereas both PI4KIIIα complexes support production of PI(4)P, the distinct functions of each complex and mechanisms underlying the distribution of PI4KIIIα between them remain unknown. In this dissertation, I present roles for differential palmitoylation patterns within a tri-Cys motif in EFR3B (Cys5/Cys7/Cys8) in controlling the distribution of PI4KIIIα between these two complexes at the plasma membrane and corresponding functions in phosphoinositide homeostasis. I used EFR3B palmitoylation mutants to investigate the palmitoylation requirement for EFR3B anchoring to the PM. I found that any two of the three cysteine residues are required for proper localization of EFR3B. Using chemical biology methods, I investigated the extent and stability of EFR3B palmitoylation and found that palmitoylation of EFR3B is highly stable, with most of the cellular pool of EFR3B being double or triple palmitoylated. The spacing of palmitoyl groups within three the doubly palmitoylated EFR3B “lipoforms” affected both its interactions with TMEM150A, a transmembrane protein governing formation of a PI4KIIIα complex functioning in rapid PI(4,5)P2 resynthesis following PLC signaling, and its partitioning within liquid-ordered and -disordered regions of the plasma membrane. Specifically, the EFR3B lipoform palmitoylated at positions C7 and C8 interacted more favorably with TMEM150A than the other lipoforms and plays a role in the formation of a PI4KIIIα dedicated to PI(4,5)P2 homeostasis in the plasma membrane. This work identifies a palmitoylation code in controlling protein–protein and protein–lipid interactions affecting a plasma membrane-resident phosphoinositide biosynthetic pathway.