Background The family of Mixed Lineage Leukaemia (MLL) histone methyltransferase proteins is often implicated in disease processes, particularly cancer. We focus on the tumour suppressors MLL3 and MLL4, which are mutated in a high percentage of cancers, but very little is known about the underlying transcriptional and epigenetic changes that contribute to malignancy. Results Here we make use of the highly accessible planarian model system to uncover a role for MLL3/4 orthologs in controlling stem cell differentiation and proliferation, suggesting conservation of tumour suppressor function over a large evolutionary timescale for these epigenetic regulators. Knockdown of the planarian Mll3/4 orthologs compromises stem cell differentiation and leads to hyper-proliferation and tumour-like outgrowth formation. The planarian system allows us to investigate the early-stage epigenetic and transcriptional changes in cells that will go on to form tumours after loss of MLL3/4 function, identifying genome wide alterations that occur early in the development of the pathology. This reveals mis-regulation of both conserved and hypothesised oncogenes and tumour suppressors, that together likely explain the cancer-like phenotype observed in planarians. Conclusions We confirm MLL3/4 tumour suppressor function and uncover a deep conservation of this role in stem cells. We find potentially conserved mis-regulated downstream targets driving the effects of MLL3/4 loss of function. Our work demonstrates the suitability of planarians for the study of epigenetic phenotypes related to cancer and stem cell function, and for capturing early causative changes in a definitive population of tumour forming stem cells in vivo.