The axon initial segment (AIS) is a specialized neuronal compartment that plays a key role in neuronal development and excitability. It concentrates multiple ion channels and cell adhesion molecules. The anchoring of these AIS membrane components is known to be highly dependent of the scaffold protein ankyrin G (ankG) but whether ankG membrane partners play a reciprocal role in ankG targeting and stabilization has not been studied yet. In cultured hippocampal neurons and cortical organotypic slices, we found that shRNA-mediated knockdown of ankG membrane partners led to a decrease of ankG concentration and perturbed the AIS formation and maintenance. These perturbations were rescued by expressing an AIS-targeted sodium channel, or a minimal construct containing the ankyrin-binding domain of Nav1.2 and a membrane anchor. We thus demonstrate that a tight and precocious association of ankG to its membrane partners is crucial for the establishment and maintenance of the AIS.