The final stages of mammalian erythropoiesis involve enucleation, membrane and proteome remodeling, and organelle clearance. Concomitantly, the erythroid membrane skeleton establishes a unique pseudohexagonal spectrin meshwork that is connected to the membrane through junctional complexes. The mechanism and signaling pathways involved in the coordination of these processes are unclear. Here we reveal an unexpected role of membrane skeleton in the modulation of proteome remodeling and organelle clearance during the final stages of erythropoiesis. We found that diaphanous-related formin mDia2 is a master regulator of the integrity of the membrane skeleton through polymerization of actin protofilament in the junctional complex. The mDia2-deficient terminal erythroid cell contained a disorganized and rigid membrane skeleton that was ineffective in detaching the extruded nucleus. In addition, the disrupted skeleton failed to activate the ESCRT-III complex, which led to a global defect in proteome remodeling, endolysosomal trafficking, and autophagic organelle clearance. We revealed that Chmp5, a component of the ESCRT-III complex, is regulated by mDia2-dependent activation of serum response factor and required for membrane remodeling and autophagosome-lysosome fusion. Mice with loss of Chmp5 in hematopoietic cells in vivo resembled the phenotypes in mDia2 knockout mice. Furthermore, over-expression of Chmp5 in mDia2 deficient hematopoietic stem and progenitor cells significantly restored terminal erythropoiesis in vivo. These findings reveal a formin-regulated signaling pathway that connects membrane skeleton to proteome remodeling, enucleation, and organelle clearance during terminal erythropoiesis.