SUMMARY The extraordinary functional range of the mature CNS requires synchronized activity of neural circuits. Formation of such circuits involves the timely specification and correct positioning of neural progenitors, and disruptions to this process are associated with neurodevelopmental disorders. Hence, a key goal of developmental neurobiology is to understand the control of neural progenitor specification in space and time in embryogenesis. One mechanism for proper spatiotemporal formation of neural progenitors involves morphogen gradients that specify distinct progenitor types at different positions. While we know that morphogens do induce different progenitor types at different positions, we do not understand how individual cells interpret morphogen signals, nor how they convert this information into distinct cell identities. An informative example of CNS morphogen action is the developing hindbrain, where retinoic acid (RA) and fibroblast growth factor (Fgf) control formation of neural compartments (rhombomeres) – each of which represents a unique progenitor population. However, we do not understand the distinct genetic programs that define individual rhombomeres, nor how they arise from the earlier hindbrain primordium. Filling these knowledge gaps is essential, but a profound barrier has been the lack of comprehensive molecular data for individual progenitors as they undergo specification in response to morphogen signals. We overcame this barrier by applying scMultiome analysis – which combines RNAseq and ATACseq of individual nuclei – to several stages of hindbrain development. At the earliest stage, we detect three populations (a.k.a., PHPDs) containing progenitors with mixed rhombomere (r) identities representing r2/r3, r4 and r5/r6 and we find that these PHPDs form in response to RA and Fgf. At later stages, our analyses – for the first time – molecularly resolve all rhombomeres and define their unique gene regulatory networks (GRNs). These advances now allow us to address several key questions: How do progenitor cells respond to morphogens? How are the mixed progenitor identities resolved into individual rhombomere identities? How are unique GRNs formed in each developing rhombomere? We will answer these questions in the context of our hypothesis that morphogens act via specific cis-regulatory elements to induce mixed identity GRNs in the PHPDs, and these are subsequently resolved via repressive genetic interactions into rhombomere-specific GRNs that specify unique progenitor types. Our project will delineate how morphogens control genetic programs for positioning and specification of neural progenitors in the hindbrain. Since morphogens control neural specification throughout the developing CNS, our findings will be broadly applicable to normal brain development, to modeling of neurodevelopmental disorders, and to the implementation of restorative or replacement strategies as clinical treatments.