Intestinal organoids and the intestinal stem cells (ISCs) that give rise to them are promising tools for disease modelling and future use in autologous stem cell therapy. However, for organoid technology to reach its full potential, there is a need to generate bona fide, mature ISCs from alternative cell sources like induced pluripotent stem cells (iPSCs) and pluripotent stem cells (PSCs) from cell banks with their various genetic backgrounds and modifications. To facilitate this, we use a predictive algorithm based on RNA sequencing data (RNAseq) and supported by chromatin state data (ATACseq), to identify key transcription factors (TFs) to direct PSC differentiation towards a desired cell state. Employing this approach, we uncovered that overexpression of Hnf4a, Vdr and Onecut2 can guide mouse embryonic stem cells differentiation towards the budding intestinal organoid state; enabling, for the first time, the generation of self-renewing ISCs from mouse PSCs. This demonstrates the feasibility of using TFs guided differentiation to generate ISCs and mature organoids from PSCs in vitro. In the human system (unlike in the mouse system), published, cytokine-driven PSC differentiation protocols exist that allow organoid differentiation, albeit with immature fetal/embryonic-like features, which impedes their potential for disease modelling. Reflective of this, RNAseq and ATACseq of human primary ISCs, PSCs and PSC-derived ISCs revealed that in vitro-derived ISCs shared some features (associated with cell cycle, metabolism and developmental state) with both PSCs and primary ISCs. Moving forward, we are testing the use of candidate TFs to shift PSC-derived human ISCs towards a fully mature state.