a,b Untransfected UCLA1 hESCs, a Control line nucleofected in parallel with the TFAP2C−/− lines, and TFAP2C−/− lines 1 and 2 are treated with 5iLAF media for 3 days (a) and 5 days (b). The TFAP2C−/− colonies show formation of morphologically distinct, flat colonies by 3 days. Scale bar indicates 100 μm. c, Top statistically enriched GO terms of genes upregulated in the TFAP2C−/− cells, calculated using a hypergeometric test with adjustment for multiple hypothesis testing33. All terms are consistent with neural differentiation of the TFAP2C−/− of cells. Terms specific to neural identity are colored blue. d, ATAC-seq peaks specific to WT cells relative to TFAP2C−/−cells after five days in 5iLAF show strong enrichment for AP2 motifs and for factors involved in pluripotency (OCT4, SOX, KLF), consistent with a loss of pluripotency in these cells. e, ATAC-seq peaks specific to TFAP2C−/− relative to WT cells after five days in 5iLAF show enrichment for motifs corresponding to such neural factors as SOX (e.g. SOX1) and ZIC (e.g. ZIC1). Motif enrichment was calculated using a cumulative binomial distribution19. f, Schematic for targeting generation of Tfap2c−/− and Tfap2a−/− Tfap2c−/− mESCs. Note that all deletions either induce frame shifts or delete splice sites. g, Western blot for Tfap2c in control and Tfap2c−/− lines. Representative of 2 independent experiments. Uncropped Western blot images are available in Supplementary Fig. 9.