The neural crest is an embryonic population of multipotent stem cells that form numerous defining features of vertebrates. Due to lack of reliable techniques to perform transcriptional profiling in intact tissues, it remains controversial whether the neural crest is a heterogeneous or homogeneous population. By coupling multiplex single molecule fluorescence in situ hybridization with machine learning algorithm based cell segmentation, we examine expression of 35 genes at single cell resolution in vivo. Unbiased hierarchical clustering reveals five spatially distinct subpopulations within the chick dorsal neural tube. Here we identify a neural crest stem cell niche that centers around the dorsal midline with high expression of neural crest genes, pluripotency factors, and lineage markers. Interestingly, neural and neural crest stem cells express distinct pluripotency signatures. This Spatial Genomic Analysis toolkit provides a straightforward approach to study quantitative multiplex gene expression in numerous biological systems, while offering insights into gene regulatory networks via synexpression analysis.