The two evolutionarily conserved mammalian lipid kinases Vps34 and PIKfyve are involved in an important physiological relationship, whereby the former produces phosphatidylinositol (PtdIns) 3P that is used as a substrate for PtdIns(3,5)P₂ synthesis by the latter. Reduced production of PtdIns(3,5)P₂ in proliferating mammalian cells is phenotypically manifested by the formation of multiple translucent cytoplasmic vacuoles, readily rescued upon exogenous delivery of PtdIns(3,5)P₂ or overproduction of PIKfyve. Although the aberrant vacuolation phenomenon has been frequently used as a sensitive functional measure of localized PtdIns(3,5)P₂ reduction, cellular factors governing the appearance of cytoplasmic vacuoles under PtdIns3P-PtdIns(3,5)P₂ loss remain elusive. To gain further mechanistic insight about the vacuolation process following PtdIns(3,5)P₂ reduction, in this study we sought for cellular mechanisms required for manifestation of the aberrant endomembrane vacuoles triggered by PIKfyve or Vps34 dysfunction. The latter was achieved by various means such as pharmacological inhibition, gene disruption or dominant-interference in several proliferating mammalian cell types. We report here that inhibition of V-ATPase with bafilomycin A1 as well as inactivation of the GTP-GDP cycle of Rab5a GTPase phenotypically rescued or completely precluded the cytoplasmic vacuolization despite the continued presence of inactivated PIKfyve or Vps34. Together, our work identifies for the first time that factors such as active V-ATPase or functional Rab5a cycle are acting coincidentally with the PtdIns(3,5)P₂ reduction in triggering formation of aberrant cytoplasmic vacuoles under PIKfyve or Vps34 dysfunction.