DNA is organised in a three-dimensional, non-random and cell-type-specific manner in the nuclear space. However, given the ubiquitous expression of structural protein complexes known to organise the DNA, it is unclear how this cell-lineage-specific architecture is established or maintained. Using cutting-edge genome-wide chromosome conformation capture techniques (in situ HiC), 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. Using knockout and conditional reintroduction systems, we show that the lineage-defining transcription factor Paired box 5 (Pax5) is critical for establishing and maintaining global genome architecture in B cells of the immune system. Interestingly, we also demonstrate that Pax5 can perform these roles independent of transcription. These results implicate sequence-specific DNA-binding proteins in global genome organisation to establish and maintain lineage fidelity.