Abstract Gene expression programs are dynamically regulated by the accessibility of chromatin for transcription factor (TF) binding, but how TFs recognize specific regulatory regions occluded by nucleosomes remains unclear. Certain TFs, termed pioneer factors, can recognize their target sites within nucleosomes, leading to the opening of chromatin. By priming cis-regulatory elements for subsequent transcriptional regulatory activity, pioneers serve as gatekeepers to cellular differentiation. Although pioneers can bind nucleosomal sites, they bind only a subset of their potential recognition sites in the genome that typically varies across cell types, thus indicating their interplay with sequence, epigenetic or other cellular features. Despite the importance of pioneer factors, what restricts pioneer binding is poorly understood. Little is known about how the sequence context of their sites in nucleosomes, the presence of histone variants or post-translational modifications (PTMs) of histones, or interactions with cofactors or chromatin readers that recognize those PTMs might influence pioneer binding to nucleosomes. No high-throughput technologies have been developed to survey the impact of these many parameters on TF pioneer binding. In this project, we will develop novel, high-throughput biochemical assays to investigate how nucleosomal sequence context, histone variants or histone PTMs influence pioneer binding of human TFs to nucleosomes. We will also investigate the interplay of pioneers, cofactors, and chromatin readers in pioneer binding. Results from these biochemical assays will be validated in vitro and used in analysis of in vivo genomic data in human cells to understand how these various features contribute to TF pioneer binding in cells. As pioneer factors play crucial roles at the top of regulatory hierarchies, these results will aid in understanding how gene regulation of cell states is encoded in the genome and the mechanisms by which it is read out.