PU.1 is an ETS-domain transcription factor that has critical roles in many aspects of haematopoiesis and immune cell fate and function. In addition, aberrant PU.1 expression has been implicated in the development of acute myeloid leukemia (AML). Loss of PU.1 during adult murine haematopoiesis results in the expansion of immature granulocytes suggesting that PU.1 plays an important role in granulocyte maturation. To understand the molecular underpinnings of this process, we performed a multi-omics analysis (transcriptomics, ChIP-seq and HiC) of the conditional deletion of PU.1 in vivo in granulocytes. We find that in contrast to wild type granulocytes, PU.1-deficient cells possessed a transcriptome of immature granulocytes, in line with their cellular phenotype. Compared to wild-type, PU.1-deficient granulocytes display altered 3D genome architecture with a significant loss of interactivity in regions bound by PU.1 in wild-type. Additionally, promoters associated with cancer and Notch signalling pathways show increased interactivity and elevated expression in PU.1-deficient cells. Analysis of publicly available acute myeloid leukaemia (AML) patient datasets revealed a subset of patients with low PU.1 expression and upregulated Notch pathway genes. This suggests a potential mechanistic link between PU.1 deficiency and Notch activation in granulocyte associated malignancy. Overall, this study establishes PU.1 as a key regulator of granulocyte maturation and lineage commitment through control of transcriptional programs and 3D chromatin architecture, with implications for cancer development.