A loss of transcription control and concomitant aberrant gene expression are hallmarks of cancer and developmental disorders. Changes in gene expression occur throughout development and require precise regulation by transcription factors and gene regulatory elements to ensure the formation of healthy tissues. However, it remains poorly understood how gradual gene expression changes during stem cell differentiation are controlled by the three-dimensional genome organization and the action of transcription factors depending on the tissue context. In the rapidly self-renewing mammalian intestinal epithelium, a directional movement of differentiating intestinal stem cells underlies their maturation to differentiated cells. Using intestinal organoids that recapitulate this behavior in vitro, I am taking a multi-pronged approach combining cutting-edge genomics with state-of-the-art quantitative live imaging methods to dissect the dynamic interplay between the 3D genome organization, cell fate-determining transcription factors and target gene expression during stem cell differentiation from the tissue to the single-molecule level.