Metabolic pathways play a critical role in driving differentiation but remain poorly understood in the development of kidney organoids. In this study, parallel metabolite and transcriptome profiling of differentiating human pluripotent stem cells (hPSCs) to multicellular renal organoids revealed key metabolic drivers of the differentiation process. In the early stage, transitioning from hPSCs to nephron progenitor cells (NPCs), both the glutamine and the alanine-aspartate-glutamate pathways changed significantly, as detected by enrichment and pathway impact analyses. Intriguingly, hPSCs maintained their ability to generate NPCs, even when deprived of both glutamine and glutamate. Surprisingly, single cell RNA-Seq analysis detected enhanced maturation and enrichment for podocytes under glutamine-deprived conditions. Together, these findings illustrate a novel role of glutamine metabolism in regulating podocyte development.