Podocyte injury and subsequent detachment are hallmarks of progressive glomerulosclerosis. In addition to cell injury, unknown mechanical forces on the injured podocyte may promote detachment. To identify the nature of these mechanical forces, we studied the dynamics of podocyte detachment using sequential ultrastructural geometry analysis by transmission electron microscopy in NEP25, a mouse model of podocytopathy induced by anti-Tac(Fv)-PE38 (LMB2), a fusion protein attached to Pseudomonas exotoxin A, targeting CD25 on podocytes. After LMB2 injection, foot process effacement occurred on day three but detachment commenced on day eight and extended to day ten, reaching toward the urinary pole in clusters. Podocyte detachment was associated with foot process effacement covering over 60% of the glomerular basement membrane length. However, approximately 25% of glomeruli with diffuse (over 80%) foot process effacement showed no detachment. Blocking glomerular filtration via unilateral ureteral obstruction resulted in diffuse foot process effacement but no pseudocysts or detachment, whereas uninephrectomy increased pseudocysts and accelerated detachment, indicating that glomerular filtrate drives podocyte detachment via pseudocyst formation as a forerunner. Additionally, more detachment was observed in juxtamedullary glomeruli than in superficial glomeruli. Thus, glomerular filtrate drives the dynamics of podocyte detachment in this model of podocytopathy. Hence, foot process effacement may be a prerequisite allowing filtrate to generate local mechanical forces that expand the subpodocyte space forming pseudocysts, promote podocyte detachment and subsequent segmental sclerosis.