The EBs were collected at Day 2, 4, and 6 after Activin A/Lithium Chloride treatment. For each time point,the cells were first treated with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher) at 37◦C for 10-15 min with pipetting. Single cell suspension was obtained by passing through 37 μM cell strainer (STEMCELL Technologies) twice. After measuring cell concentration, approximately 1 million of cells were centrifuged at 300 rcf for 5 min. Nuclei were isolated by following the protocol provided by 10x Genomics (Nuclei isolation for single cell multiome ATAC + Gene expression sequencing, CG00365, Rev A). The final nuclei concentration was adjusted to 3000 cell/μL in 1X Nuclei Buffer (10x Genomics). The sample was immediately submitted to Stanford Genomics Service Center (SGSC) for single cell sorting using 10x Chromium Controller (target cells: 5000 per replicate, total 2-3 replicates per time point). The singe cell multiome library was generated using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle Kit (10x Genomics, PN-1000283). Single cell multiome