Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells for application in disease modeling, diagnostics, drug screening and cell-based therapies for Parkinson's disease. An increased understanding of the timing and molecular mechanisms that promote the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from pluripotent stem cells. Here, we use droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11 weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach reveals that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence, they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons that can be used to guide stem cell-based therapies and disease modeling approaches in Parkinson's disease.