Gene expression requires the targeting of transcription factors (TFs) to regulatory sequences often occluded within nucleosomes. To comprehensively examine TF nucleosome binding, we developed Pioneer-Seq. In Pioneer-seq a library of thousands of nucleosomes are formed from sequences containing a TF binding site (TFBS) variant in all possible nucleosome orientations and within the linker regions. Pioneer-seq has the unique ability to simultaneously examine nucleosomes created with various nucleosome positioning sequences and examine binding to in vivo targeted nucleosomes (ITNs). Pioneer-seq can be applied to address various mechanistic models for TF-nucleosome binding directly and can be used to uncover inherent TF-interaction differences. To demonstrate Pioneer-seq, we examined nucleosome binding by OCT4, SOX2, KLF4, and c-MYC. Our results demonstrate that all studied TFs can bind at nucleosome edges and nucleosome sequence is the primary factor regulating TF binding. In addition, KLF4 can bind to a non-canonical TFBS located 20 bp from the nucleosome dyad. Examination of ITNs showed binding differences between the TFs, with KLF4 and SOX2 binding more often near nucleosome centers. Overall, our results demonstrate differences in how TF recognizes their TFBS within a nucleosome and begins to define the mechanistic requirements for pioneer factor binding.