Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84~97 events of 3D diffusion (3.3~3.7s) interspersed with brief non-specific collisions (0.75~0.9s) before acquiring and dwelling at specific target DNA (12.0~14.6s). Sox2 employs a 3D diffusion-dominated (~80 % of time) search mode facilitated by 1D sliding along open DNA to efficiently seek for targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine tuning TF dynamics and influence of the epigenome on TF search parameters.