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Freshwater planarian flatworms have always fascinated scientists with their ability to regenerate missing body pieces and to maintain tissue homeostasis. This developmental plasticity is due to a population of pluripotent adult stem cells, also called neoblasts. Using the planarian model organism Schmidtea mediterranea, we aim to understand the mechanisms controlling regeneration and neoblast homeostasis. Stem cell studies from other organisms showed that pluripotency can be controlled by epigenetic mechanisms, such as DNA methylation and histone modifications, by affecting gene expression. In S. mediterranea, the gene encoding a Methyl-CpG Binding Domain (MBD) protein ortholog, Smed-mbd213, was identified. Smed-mbd213 is homologous to the vertebrate MBD2 and MBD3, with the latter being found to affect embryonic stem cell differentiation in mammals. In S. mediterranea Smed-mbd213 is expressed in neoblasts, and its knockdown by RNA interference (RNA i) affected both regeneration and tissue homeostasis. Amputated Smed-mbd213(RNAi} animals formed blastema but failed to correctly regenerate the missing structures such as the brain, photoreceptors and the pharynx, and intact Smedmbd213( RNAi) animals gradually regressed tissue. Using markers for neoblasts and their early and late progeny cells on Smed-mbd213(RNAi) intact and regenerating animals, we found that Smed-mbd213 is not required for stem sell maintenance or the formation of some subpopulations of early progeny cells, which in fact accumulated. Smed-mbd213 is actually required for the formation of late progeny cells, representing the first evidence of a neoblast gene that is required for the differentiation of late but not early progeny cells. In addition, Smed-MBD2/3 is the first invertebrate member of the MBD protein family found to be required for stem cell differentiation similar to the mammalian MBD3. In the search for DNA methylation components in S. mediterranea genome, a DNA methyl transferase candidate gene, Smed-Dnmt2, was found, but knockdown experiments did not show any vital role of this gene in the regeneration process. Also, we did not fmd evidence for cytosine methylation in S. mediterranea genome. This means that neoblast ( pluripotency is not controlled by this epigenetic modification and that Smed-MBD2/3 functions independently of cytosine methylation. Finally, to determine more players in the neoblast differentiation process, we analysed the transcriptome of Smed-mbd213(RNAi) animals and performed an RNAi screen on a group of selected transcripts, We found a novel gene called myst-related that is expected to expresses a chromatin modifying protein. Its knockdown by RNAi gave a similar but more severe phenotype than that of Smed-mbd213, affecting tissue differentiation during regeneration and homeostasis. Whether Smed-mbd213 and myst-related function in the same or different pathways, our study presents new effectors of neoblast differentiation that possibly act by modifying the chromatin architecture. ii