<p>The field of human induced pluripotent stem cells (hiPSCs) has seen significant progress since the discovery of reprogramming somatic cells using the transcription factors Oct4, Sox2, Klf4, and c-Myc. hiPSCs are similar to embryonic stem cells in a primed state of pluripotency and have the potential to differentiate into any adult human cell type, offering a versatile tool for research and potential therapeutic applications. However, the efficiency of differentiation protocols for generating complex structures with multiple cell types, Like kidney organoids, remains a challenge. This study investigates the impact of treating hiPSCs with a low-dose dimethyl sulfoxide to enhance kidney organoid differentiation using the stepwise 2D monolayer-based protocol developed by Morizane et al. 2017. We found that treating hiPSCs with 1–2% DMSO affects gene expression of pluripotent transcription factors, the epigenetic landscape, and hiPSC colony morphology. Our findings also suggest DMSO treatment enhances the expression of the key metanephric mesenchyme nephron progenitor marker, SIX2 after 9 days of kidney organoid differentiation and helps improve hiPSC differentiation protocol efficiency toward the development of tubular kidney organoids. Further research is needed to fully elucidate the mechanisms underlying these effects and refine the differentiation process for potential in vitro research applications in biomedical research and drug development.</p> Graphical Abstract <p></p>