We have previously described the generation of floating, 3D neural cultures from hPSCs resembling the pallium (hCS) that contain deep and superficial layer cortical glutamatergic neurons, as well as astrocytes11. To specify spheroids resembling the ventral forebrain or the subpallium (hSS), we exposed early spheroids that were patterned by double SMAD inhibition to small molecules modulating the WNT and SHH pathways in the presence of the growth factors FGF2 and EGF (Fig. 1a; Supplementary Table 1). At day 25 of hSS in vitro differentiation, we observed a strong induction of the transcription factor NKX2-1 in hSS accompanied by high levels of FOXG1 expression and down-regulation of the pallial marker EMX1, suggestive of a subpallial fate (Fig. 1b; Supplementary Table 2). We next examined the hSS cytoarchitecture in cryosections and noticed that NKX2-1 was expressed in ventricular zone (VZ)-like structures at day 25 (Fig. 1c) but was distributed more broadly at later stages (Fig. 1d). At day 60, we observed strong expression of GABA and the GABA-synthesizing enzyme GAD67 in neurons (Fig. 1e, f). Of the known markers that identify GABAergic
