By directing the differentiation of human pluripotent stem cells (hPSCs) and supporting their intrinsic ability to self-organize in three-dimensional (3D) space, researchers have successfully generated cerebral organoids comprised of cell types specific to sub- regions of the brain; however, a protocol which enriches organoids with mature granule neurons of the human hippocampus has yet to be developed. Here, we investigate the culture conditions and developmental pathway regulation requirements for the generation of dentate gyrus (DG), hippocampal-like organoids. The DG is one of the few sites in the mammalian brain known to support adult neurogenesis, and this phenomenon might be linked to the DG’s function of new and episodic memory formation and its implication in neuropsychiatric disorders including depression and schizophrenia. We first established a protocol for robust brain organoid generation from human embryonic stem cells (hESCs) by investigating the effects that early PSC maintenance conditions would have on later organoid generation and development. By employing these optimized organoid generation protocols, we were able to screen various enhancers and inhibitors of developmental signaling pathways implicated in vertebrate DG development and those identified from established monolayer hPSC differentiation protocols for obtaining granule neurons and other associated DG cell types. A set of maturation factors was also tested in different combinations and at various timepoints to promote the terminal differentiation of neural progenitor cells (NPCs) to granule neurons and to support granule neuron maintenance. Organoids developed by these methods were analyzed at several timepoints during development and were screened for the presence of prospero homeobox protein 1 (PROX1) expressing granule neurons through immunohistochemical analysis. Additionally, immunostaining was performed for markers associated with telencephalic NPCs thought to give rise to mature granule-like neurons. Using these strategies, we have set the stage for the generation of DG-like organoids in vitro.