For microarray analysis of ESC-derived hematopoietic progenitors, co-cultures were performed as described above. On Day 9, 23 hours after transfer from OP9-C to OP9-DL1 stroma, CD45+ cells were collected by FACS. RNA was isolated using the Qiagen RNAeasy columns, as per the manufacturer’s instructions. Double-stranded cDNA was prepared from ~1 μg of total RNA using the Affymetrix cDNA synthesis kit (Affymetrix, Santa Clara, Ca), and a single round of in vitro transcription was performed using an IVT labeling kit (Affymetrix). cRNA product was purified using a GeneChip Sample Cleanup Module (Affymetrix) and 20 μg of biotin-labeled cRNA was fragmented and hybridized to Affymetrix Mouse MOE430_2.0 microarrays for 16 hr in the Affy 640 hybridization oven at a speed of 60 rpm. Microarrays were washed and stained using and Affymetrix FS400 Fluidics Station. GeneChip arrays were scanned using a GeneChip Scanner 3000 (Affymetrix). Initial probe set intensities were quantified using the GeneChip Operating Software (GCOS). All hybridized chips exceeded standard quality control criteria as recommended by the manufacturer, and as previously described (48 (link), 49 (link)). Microarray data are available from the Gene Expression Omnibus under accession number GSE199279 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE199279).