Phosphatidylinositol 4 kinase III (PI4KIII/PI4KA) is an essential lipid kinase that is critical for regulating plasma membrane identity. PI4KA is primarily recruited to the plasma membrane through targeted recruitment by the proteins EFR3A and EFR3B, with these binding to the PI4KA accessory proteins TTC7 (TTC7A and TTC7B) and FAM126 (FAM126A and FAM126B). Here we characterised how both EFR3 isoforms interact with all possible TTC7 and FAM126 combinations and developed a nanobody that specifically blocks EFR3 mediated PI4KA recruitment in TTC7B containing complexes. Using a yeast display approach, we generated a nanobody that is selective for TTC7B and blocks EFR3 binding. Cryo-electron microscopy and hydrogen deuterium exchange mass spectrometry reveal that the nanobody sterically blocks EFR3 recruitment and shows an extended interface with both PI4KA and TTC7B. Overall, this work provides insight into PI4KA regulation and is a useful tool for manipulating unique complexes of PI4KA that may be valuable for future therapeutic targeting of PI4KA.