All somatic cells in an organism contain the same genetic material yet achieve distinct morphologies and functions. This process of cellular differentiation is guided by transcription factors (TF) binding DNA to orchestrate the coordinated activation of genes. First, I investigated this process as initiated by the action of a Pioneer TF which specifically can access DNA in silent chromatin to control lineage specific transcriptional programs. The transcription factor TCF-1 has been previously described as capable of interacting with repressed chromatin and cementing T cell identity. However, it remained unclear how regions outside of TCF-1’s canonical DNA-binding domain contribute to this function. I discovered that an intrinsically disordered region within the N-terminus of TCF-1 (termed L1) was integral for T cell lineage fidelity enabling TCF-1 to target silent chromatin and repress alternative lineages. The L1 domain could be replaced with a heterologous disordered domain from another TF to restore both binding and T cell development. Second, I examined how TFs facilitate the spatial assembly of enhancers and promoters within the nucleus through coordination of the 3D organization of DNA. We found TCF-1 was required for the dissolution of topologically associated domains to merge insulated regions at T cell enhancers. This corresponded to TCF-1 dependent deposition of the cohesin loading protein NIPBL to maintain expression of T cell genes. Finally, I examined how single nucleotide polymorphisms (SNPs), representing genetic variation between individuals in a species, can disrupt transcriptional regulation leading to common diseases. We found pathogenic genetic variation in the Non-Obese Diabetic (NOD/ShiLtJ) mouse, a model of type I diabetes, was linked to the genome re-organization in T cells downstream of strain specific binding of architectural proteins. This resulted in the upregulation of Krüppel-associated box zinc finger protein (KRAB-ZFP) genes a feature that was conserved in human immune cell infiltrates isolated from the pancreas. Together these findings suggest a model through which lineage defining transcription factors direct cell fate decisions and mediate the dissolution of repressive chromatin barriers through genome re-organization. This genome re-organization underscores normal development but also can contribute to complex immune-mediated diseases.