ABSTRACT Background: The proximal tubule plays a major role in electrolyte homeostasis. Previous studies have shown that HNF4A regulates reabsorption-related genes and promotes proximal tubule differentiation during murine kidney development. However, the functions and gene regulatory mechanisms of HNF4 family genes in human nephrogenesis have not yet been investigated. Methods: We generated HNF4A-knock out (KO), HNF4G-KO, and HNF4A/4G-double KO human pluripotent stem cell (PSC) lines, differentiated each into kidney organoids, and used immunofluorescence analysis, electron microscopy, and RNA-seq to analyze them. We probed HNF4A binding sites genome-wide by cleavage under targets and release using nuclease (CUT&RUN) sequencing in both human adult kidneys and kidney organoid-derived proximal tubular cells. Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation (CRISPRa) validated HNF4A and HNF4G function in proximal tubules during kidney organoid differentiation. Results: Organoids lacking HNF4A, but not HNF4G, showed reduced expression of transport-, endocytosis-, and brush border-related genes, as well as disorganized brush border structure in the apical lumen of the organoid proximal tubule. CUT&RUN revealed that HNF4A primarily bound promoters and enhancers of genes that were down-regulated in HNF4A-KO, suggesting direct regulation. Induced expression of HNF4A or HNF4G by CRISPRa drove increased expression of selected target genes during kidney organoid differentiation. Conclusions: Our study reveals regulatory mechanisms of HNF4A and HNF4G during human proximal tubule differentiation. The experimental strategy can be applied more broadly to investigate transcriptional regulation in human kidney development.