Genome-wide transcriptional activity involves the binding of many transcription factors (TFs) to thousands of sites in the genome. Pioneer TFs are a class of TFs that maintain open chromatin and allow non-pioneer TFs access to their target sites. Determining which TF binding sites directly drive transcription remains a challenge. Here, we use acute protein depletion of the pioneer TF SOX2 to establish its functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of the pioneer factor. To understand the relationship with transcription, we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to all other SOX2 binding sites. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted mainly at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.