Achieving robust and high levels of NKX6.1 expression in human pluripotent stem cell-derived pancreatic progenitor cells has been a significant challenge in the field. As demonstrated in the 2013 publication from Dr. Tim Kieffer’s Lab at the University of British Columbia, in collaboration with BetaLogics (Rezania et al., 2013), enriching NKX6.1 expression in differentiating pancreatic progenitor cells significantly improves their ability to mature in vivo towards functional endocrine cells. Since this publication, more emphasis has been placed on characterizing PDX-1 and NKX6.1 co-expression during the differentiation process. We routinely assess the formation of pancreatic progenitor cells from human pluripotent stem cells by examining the expression of PDX-1 and NKX6.1 by qPCR as well as the co-localization of these markers by flow cytometry. Looking at a sample set of seven independent differentiation experiments, we find that the percentage of cells expressing PDX-1 as measured by flow cytometry is 80.7 ± 10.2% (mean ± SD) while the percentage of cells expressing NKX6.1 is 76.1 ± 6.3%. Within this data set, we see similarly low variation in the expression of NKX6.1 as with PDX-1. Nearly all PDX-1-positive cells also express NKX6.1, indicating a very efficient formation of pancreatic progenitor cells when using the STEMdiff Pancreatic Progenitor Kit. In addition, an examination of gene expression by qPCR indicates upregulation of PDX-1 and NKX6.1 to similar levels by the end of Stage 4. In five experiments, we found the upregulation of PDX-1 to be 2.7 ± 1.1-fold (mean ± SD) compared to housekeeping genes TBP and 18S ribosomal RNA and the upregulation of NKX6.1 to be 3.3 ± 1.2-fold. Both genes were upregulated to a similar extent with similarly low standard deviations, again demonstrating the reproducibility of generating pancreatic progenitor cells using this kit.