Oct4 and Sox2 are two essential transcription factors that co-regulate target genes for the maintenance of pluripotency. However, it is unclear whether they interact prior to DNA binding or how the target sites are accessed in the nucleus. By generating fluorescent protein fusions of Oct4 and Sox2 that are functionally capable of producing iPSCs (induced pluripotent stem cells), we show that their interaction is dependent on the presence of cognate DNA-binding elements, based on diffusion time, complex formation and lifetime measurements. Through fluorescence correlation spectroscopy, the levels of Oct4 and Sox2 in the iPSCs were quantified in live cells and two diffusion coefficients, corresponding to free and loosely bound forms of the protein, were distinguished. Notably, the fraction of slow-diffusing molecules in the iPSCs was found to be elevated, similar to the profile in embryonic stem cells, probably due to a change in the nuclear milieu during reprogramming. Taken together, these findings have defined quantitatively the amount of proteins pertinent to the pluripotent state and revealed increased accessibility to the underlying DNA as a mechanism for Oct4 and Sox2 to find their target binding sites and interact, without prior formation of heterodimer complexes.