The ability to differentiate human pluripotent stem cells (hPSCs) into lung cells offers enormous research and therapeutic potential for respiratory diseases. However, protocols for generating functional lung tissue from hPSCs are inefficient and highly variable. Our central hypothesis is that cell-to-cell variability and spatial organization impact the final output of hPSC differentiation. We are applying single-cell RNA sequencing and imaging techniques to understand how these mechanisms affect cell fate decisions during definitive endoderm specification, the first critical step towards lung commitment. Our first data set was generated from a differentiation time course that spanned 4 days (hPSCs to definitive endoderm) using the Fluidigm system. As we begin to study subsequent differentiation time points (e.g. anterior foregut endoderm) to address increasing population heterogeneity we will use InDrops technology developed by Dr. Allon Klein.