SUMMARY Despite the apparent uniform cytoarchitecture of the cerebellar cortex, imaging studies have revealed discrete functional domains within it. We have recently identified numerous Purkinje cell (PC) subtypes that occupy discrete domains of the developing mouse cerebellar cortex, providing a basis for the functional parcellation of the cerebellar cortex. Climbing fibers, which originate from individual inferior olive (IO) nuclei, specifically target PCs within different domains of the cerebellar cortex. A significant knowledge gap exists regarding the molecular heterogeneity of IO neurons and the mechanisms governing their diversification. Preliminary data suggest the crucial role of transcription factor Foxp2 in diversifying PCs and IO neurons, leading us to hypothesize analogous heterogeneity in climbing fiber neurons. To test this hypothesis, we propose an innovative strategy integrating single-cell RNA-seq and single-cell resolved spatial protein profiling to develop a three-dimensional molecular atlas of the developing IO, under normal conditions and in the absence of Foxp2. Aim 1 is centered on constructing a 3D molecular atlas of the developing IO, while Aim 2 investigates the impact of Foxp2 loss on IO development. We anticipate that the completion of these aims will yield a detailed spatial molecular atlas of the IO and will provide insights into the molecular regulation of IO nucleus formation by Foxp2. This research will not only bridge the existing knowledge gap regarding IO neurons but also enhance our understanding of the discrete connectivity and function of different domains within the cerebellar cortex.