Stem cell maintenance and differentiation programs are directed by transcription, which involves extensive protein-protein interactions between master transcription factors, the Mediator complex, and RNA polymerase II (Pol II). The Mediator complex aids transcription factors to recruit Pol II to target promoters and is critical for activating all eukaryotic protein-coding genes. Although Mediator and Pol II are known to interact, the real-time diffusivity, stability, and kinetics of these interactions in living cells remain unknown. I aim to utilize single-molecule microscopy to study Mediator-Pol II interaction dynamics in the context of Mediator-related neurodevelopmental disorders. The results of this study will provide a spatiotemporal understanding of the molecular mechanisms and key signaling pathways that regulate early neurogenesis and identify possible therapeutic avenues for Mediator-related pathologies.