Acknowledgements: We would like to acknowledge Birgit Campbell, Christina Scherb, and Marco Crovella for their technical assistance, Gabrielle Lederer (Cytogenetic Department, TUM) for karyotyping, Dr. Rupert Öllinger (TUM, Germany) for sequencing, Dr. David Elliott for sharing the ES03 and ES03-NKX2.5eGFP cell lines, Drs. Ed Stanley and Andrew Elefanty (MCRI, Australia) for advice in construct design and gene targeting, and Dr. Sasha Mendjan for advice and discussion. This work was supported by the European Research Council (ERC) (grant 788381 to A.Mo. and grant 261053 to K-.L.L.), the Else-Kroener-Fresenius Stiftung (EKFS, to A.G.), the German Research Foundation (grant GO3220/1-1 to A.G.; Transregio Research Unit 152 to A.Mo. and K-.L.L.; Transregio Research Unit 267 to A.Mo., K-.L.L., and P.G.), the German Centre for Cardiovascular Research (DZHK) (grant FKZ 81Z0600601 to A.Mo. and K-.L.L.; grant 81X3600607 to J.K.), the Fondazione Umberto Veronesi (to G.S.).Funder: German Centre for Cardiovascular ReserachFunder: Else Kröner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation); doi: https://doi.org/10.13039/501100003042Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease
