Histone deacetylase inhibitors are a class of drugs with demonstrated activity in haematological malignancies. Vorinostat and romidepsin are FDA approved for treatment of cutaneous T-cell lymphoma and inhibit HDACs1,2,3 and 6 or HDAC1,2 and 3, respectively. HDAC-selective inhibitors could improve exposure and dose levels in vivo, ultimately improving efficacy and reducing toxicity in the clinic. We utilised RNAi technology to investigate whether loss of individual HDACs could phenocopy the effects of HDACi in Eµ-myc B-cell lymphoma and acute myeloid leukaemia (AML). Murine HDACs were individually knocked down in Eµ-myc (HDACs1,2,3,6) and AML (MLL-AF9;Nras; HDACs1-11) cells using constitutive (pLMS/pLMN) or Tetracycline-inducible (pTRMPVIN) vectors in vitro and in vivo. Cell proliferation, apoptosis, cell cycle, protein expression and gene knockdown were assessed by FACS or Western blot. Gene expression was assessed by RNAseq. Loss of HDACs1, 2 or 6 had no long term effects on cell growth, while cells lacking HDAC3 were reproducibly lost from long term culture. This phenotype was not prevented by Bcl-2 over-expression, caspase inhibition or knockout of p21 but appeared dependent on the expression of p53, including specific mutants of p53. HDAC3 knockdown altered the transcription of <0.05% genes in Eµ-myc cells. Importantly, loss of HDAC3 in vivo reduced the growth rate of both Eµ-myc lymphoma and AML. These results demonstrate the importance of HDAC3 in the proliferation of Eµ-myc lymphoma and AML. Importantly, loss of HDAC3 reduces tumour cell proliferation and partially phenocopies the effects of pan-HDACi. This suggests that HDAC3-specific inhibitors might prove useful for the treatment of haematological malignancies, however only when p53 is present. Further work investigating the molecular events that underpin the loss of proliferation induced by HDAC3 knockdown/inhibition, particularly those relating to p53 (acetylation, specific mutants), are currently underway.