Cooperative binding of transcription factors (TFs) to chromatin orchestrates eukaryotic gene expression and cell fate specification. A subset of TFs, known as pioneer factors (PFs), possess an inherent ability to target DNA sites occluded by nucleosomes. Due to limited temporal resolution of genomic and structural methods, the kinetics of PF-chromatin interaction remain poorly characterized. To this end, we use single-molecule multicolor fluorescence microscopy to image individual PF binding events on DNA and nucleosome substrates in real time, focusing on Oct4 and Sox2, two prominent Yamanaka factors. We find that Oct4 and Sox2 exhibit distinct properties of nucleosome targeting where Oct4 indiscriminately binds to all nucleosomal DNA positions, while Sox2 binds preferentially to the dyad axis. We further show Oct4 and Sox2 are hierarchically recruited to the nucleosome and display nonreciprocal cooperativity with Oct4 regulating Sox2’s pioneer activity but not vice versa. Genome-wide analysis performed by the Zhao Lab recapitulates the positional preference of Oct4 and Sox2 binding sites relative to nucleosome positions. This study clarifies the biophysical rules governing the Sox2-Oct4 partnership and suggests that the same set of TFs can differentially regulate gene activities based on their motif positioning in the nucleosomal context.