Cytomegaloviruses (CMVs) initiate the stepwise establishment of their cytoplasmic virion assembly compartment (cVAC) at the earliest stages of infection by expanding the interface between early endosomes (EEs) and endosomal recycling compartment (ERC) and relocating the Golgi complex. This membranous organelle reorganization involves phosphatidylinositol-3-phosphate (PI(3)P)-containing membrane domains, as PI(3)P is the master regulator of the EE pathway. Our goal was to investigate the contribution of PI(3)P-dependent EE domains to cVAC biogenesis and their role in the consequent progression of the CMV life cycle. Our functional analysis of PI(3)P was based on rapid depletion of PI(3)P production at EE membranes using VPS34-IN1, a specific and reversible inhibitor of Vps34 component of class III PI(3)P kinase, and on saturation of PI(3)P by overexpression of recombinant PI(3)P-binding domains (2xFYVE, p40PX). PI(3)P depletion led to rapid reorganization of the Rab11a-dependent pathway of the ERC, and abolished recycling cargo sorting in this pathway as well as membrane flux from EE to the ERC. Despite these alterations and although PI(3)P+ and Vps34+ endosomes build a substantial part of cVAC, the PI(3)P inhibition did not prevent the establishment of infection, rogression through the early phase of infection, and membrane reorganization associated with cVAC development, as analyzed by confocal imaging and Western blot analysis. However, the drastically reduced viral growth under PI(3)P depletion and the significant inhibition of late infection events (DNA replication, late protein expression) together with the reduced release of infectious virions suggest that PI(3)P is essential for entry into the late phase of infection and the final formation of progeny virions.