A complex set of extrinsic and intrinsic signals acts in specific temporal and spatial orders to enable neural differentiation during development. These processes have been extensively studied in animal models, but human neural development remains much less understood. This lack of detailed information about human early neurogenesis is a hindrance for the differentiation of pluripotent stem cell lines into specific neuronal phenotypes. Therefore, it is important to strengthen the interspecies comparative approaches. We describe a novel model system in which in vitro differentiation of human and mouse embryonic stem (ES) cells are temporally aligned to each other and compared with mouse telencephalic neurogenesis in vivo. In this comparative model system, we tested the in vitro role of Hedgehog (Hh) signaling for ES cell-derived telencephalic differentiation. In vivo, Hh signaling mediates dorsal/ventral patterning during early stages of telencephalic development. We monitored the effect of pharmacological modulators of the Hh signaling pathway, purmorphamine-an agonist and cyclopamine-an antagonist of the Smoothened receptor (Smo), on the expression of region-specific transcription factors and signaling molecules relevant for telencephalic development in vivo. Purmorphamine strongly upregulated the expression of telencephalic ventral markers Nkx2.1, Nkx6.2, Lhx6, and Lhx8 in mouse and human cells, thus reflecting the in vivo process of the medial ganglionic eminence patterning and specification. Cyclopamine upregulated the expression of telencephalic dorsal markers, but at lower levels in human compared with mouse cells. Modulation of Smo in vitro differentially affected, in mouse and human cells, the expression of molecules of the Hh pathway, especially the Gli1 and Gli3 effectors, Sonic Hh ligand and Ptch receptors. These results provide evidence for the different default differentiation of mouse and human ES cells and prove the utility of the comparative system for optimizing the directed differentiation of human pluripotent stem cells.