Transcription factors (TFs) bind to DNA to control gene expression, thereby regulating crucial cellular processes such as pluripotency maintenance and differentiation. How TFs move and interact with DNA across time and space remains understudied, despite the fact that these dynamics ultimately influence a cell’s response to differentiation cues. Robust pipelines are therefore needed to track TF dynamics in live cells and embryos. To address this, we established a state-of-the-art, high-density 3D single-molecule localisation microscopy approach that captures the distribution and dynamics of TFs across the entire nucleus both in vitro and in vivo. We also developed advanced analysis pipelines that extract key biophysical parameters, including diffusion rate, exploration radius, DNA residence time, and spatial clustering metrics, which together provide novel insight into TF spatial organisation. We applied this approach to investigate SOX2 and NANOG spatiotemporal dynamics in naïve pluripotent stem cells. Although both TFs exhibited identical fractions of DNA-bound populations, NANOG compensated for its lower expression levels by binding DNA longer, while SOX2 achieved similar DNA occupancy through higher expression levels even though its binding times were shorter. Despite their similar size and shared role in pluripotency maintenance, SOX2 and NANOG exhibited distinct diffusion modes but comparable exploration radii, pointing to unique intrinsic regulatory mechanisms, such as NANOG’s ability to engage in protein–protein interactions. Moreover, our cluster analysis revealed that both NANOG and SOX2 form clusters of DNA-bound and freely diffusing molecules, suggesting a shared spatial organisation despite distinct DNA-binding dynamics. To probe how NANOG’s intrinsic regulatory mechanisms shape its unique spatiotemporal behaviour, we disrupted its ability to oligomerise or form protein–protein interactions, and revealed how this governs DNA binding, diffusion, and spatial clustering. Finally, our robust pipeline provides a means to elucidate the spatiotemporal dynamics of other TFs, establishing it as a powerful tool for studying TF biophysics in diverse cellular processes.