show Abstracthide AbstractHaematopoietic stem cells (HSCs) have long been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the FAcilitates Chromatin Transcription (FACT) complex, is required for HSC formation. Zebrafish supt16h mutants express reduced levels of Notch signalling components, genes essential for HSC development, due to abrogated transcription. Classically, Supt16h regulates transcription and nucleosome reorganization. Whereas global chromatin accessibility in supt16h mutants is unaffected, we observe a specific increase in accessibility at the p53 locus, causing an accumulation of p53 mRNA and protein. We further demonstrate that P53 levels directly influence expression of the Polycomb Group protein, phc1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues both loss of Notch and loss of HSC phenotypes in supt16h mutants. Taken together, our results highlight a previously uncharacterized relationship between supt16h, p53, and phc1 to specify HSCs via modulation of Notch signalling. Overall design: RNA-sequencing was performed on 32hpf wildtype (WT) and supt16h-/- whole embryos to examine differential expression data. 40 embryos were collected for each replicate.