Significance Statement Epigenetic changes have been proposed to mediate nephron endowment during development, a critical determinant of future renal disease development. Hydroxymethyl cytosine, an epigenetic modification important for gene regulation, is abundant in the human kidney, but its physiologic role and the role of DNA demethylase enzymes encoded by the Tet1, Tet2, or Tet3, which mediate cytosine hydroxymethylation, are unclear. By genetically deleting Tet1, Tet2, or Tet3 in nephron progenitors in mice, the authors showed that combined Tet2 and Tet3 loss in nephron progenitors cause defective kidney development, leading to kidney failure and perinatal death. Tet2 and Tet3 deletion also caused an alteration in demethylation and expression of genes critical for nephron formation. These findings establish that Tet2- and Tet3-mediated cytosine hydroxymethylation in nephron progenitors plays a critical role in nephron endowment. Background Nephron endowment is a key determinant of hypertension and renal disease in later life. Epigenetic changes have been proposed to mediate fetal programming and nephron number. DNA cytosine methylation, which plays a critical role in gene regulation, is affected by proteins encoded by the ten-eleven translocation (TET) DNA demethylase gene family (Tet1, Tet2, and Tet3), but the roles of TET proteins in kidney development and nephron endowment have not been characterized. Methods To study whether epigenetic changes—specifically, active DNA hydroxymethylation mediated by Tet1, Tet2, and Tet3—are necessary for nephron progenitor differentiation and nephron endowment, we generated mice with deletion of Tet1, Tet2, or Tet3 in Six2-positive nephron progenitors cells (NPCs). We then performed unbiased omics profiling, including whole-genome bisulfite sequencing on isolated Six2-positive NPCs and single-cell RNA sequencing on kidneys from newborn mice. Results We did not observe changes in kidney development or function in mice with NPC-specific deletion of Tet1, Tet2, Tet3 or Tet1/Tet2, or Tet1/Tet3. On the other hand, mice with combined Tet2 and Tet3 loss in Six2-positive NPCs failed to form nephrons, leading to kidney failure and perinatal death. Tet2 and Tet3 loss in Six2-positive NPCs resulted in defective mesenchymal to epithelial transition and renal vesicle differentiation. Whole-genome bisulfite sequencing, single-cell RNA sequencing, and gene and protein expression analysis identified a defect in expression in multiple genes, including the WNT-β-catenin signaling pathway, due to a failure in demethylation of these loci in the absence of Tet2 and Tet3. Conclusions These findings suggest that Tet2- and Tet3-mediated active cytosine hydroxymethylation in NPCs play a key role in kidney development and nephron endowment.