hESCs were dissociated to single cells and plated on Matrigel or Synthemax II‐SC Substrate (Corning, USA, https://www.corning.com) coated plates at a density of 52.6 K/cm2 in mTeSR1 with 5 µM blebbistatin, a time point designated as day minus 1 (d‐1). Unless otherwise specified, a Matrigel cover layer was not added to the cultures after plating. One day after plating, mTeSR1 was completely exchanged for N2B27 media [1:1 mix of DMEM/F12 and Neurobasal with 1× GlutaMAX Supplement, 1× antibiotic‐antimycotic, 1% N2 Supplement, and 2% B27 Supplement (all from ThermoFisher Scientific)] to start differentiation; this day was designated as day 0 (d0). Small molecules were added to the cells on day 1 (d1), 24 hours after d0. Small molecule addition was done in fresh N2B27 media. Cells were fed with a full exchange of N2B27 media every other day unless a small molecule was to be removed or added on that day of differentiation, requiring daily feeding. The following small molecules were aliquoted as 1,000× stocks in dimethyl sulfoxide (DMSO) and used at the working concentration noted in parentheses: Forskolin (FSK; 25 µM—Cell Signaling Technology, Danvers, MA, https://www.cellsignal.com), Dorsomorphin (1 µM—R&D Systems, Minneapolis, MN, https://www.rndsystems.com), IDE2 (2.5 µM—R&D Systems), DAPT (10 µM—Cell Signaling Technology), LDN‐193189 (0.5 µM—Stemgent, Lexington, MA, https://www.stemgent.com), SB431542 (10 µM—Sigma‐Aldrich). Nicotinamide (NIC; Sigma‐Aldrich) was resuspended in water at 100× and used at a 10 mM working concentration. Noggin (ThermoFisher Scientific) was resuspended in 10 mM acetic acid with 0.5% bovine serum albumin (BSA) for a 1,000× stock and used at 100 ng/ml. All small molecules were added as indicated. Specifically, for our DIDNF+D protocol, Dorsomorphin and IDE2 (DID) were added from day 1 to 6, NIC from day 1 to 10, (FSK from day 1 to 30, and DAPT from day 18 to 30. Differentiation was carried out at 37°C in 5%CO2/20% O2. DIDNF+D = Dorsomorphin+IDE2+Nicotinamide+Forskolin+DAPT