Every nucleus in your body contains approximately two metres of DNA. This DNA is fantastically organised in the nuclear space in a three-dimensional, non-random and cell type specific manner. However, given the ubiquitous expression of structural protein complexes known to organise the DNA, it is unclear how cell lineage-specific architecture is established or maintained. Using cutting-edge genome-wide chromatin conformation capture techniques, we have demonstrated that in addition to their canonical function of simply turning genes ‘on’ or ‘off’, lineage-defining transcription factors regulate three-dimensional genome organisation. We show that different immune cell populations display distinct global genome architecture that underlies their transcriptional programs and establish that the lineage-defining transcription factor Paired box 5 (Pax5) is critical for establishing and maintaining this architecture in B cells. These results implicate sequence-specific DNA-binding proteins in global genome organisation to establish and maintain lineage fidelity.