Hematopoietic stem cells (HSCs) are a powerful resource for both regenerative medicine and the study of human developmental biology. Though much is known about HSC physiology and development in mice, experimental limitations make their characterization a greater challenge in human. As such, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are currently the best systems with which to model early human hematopoietic development in vitro, thus providing insight regarding crucial factors delineating HSC emergence, maintenance and subsequent differentiation. However, generation of HSCs from hESCs and iPSCs relies on an intimate understanding of both the in vivo hematopoietic microenvironment as well as HSC phenotype for their prospective isolation. Though current in vitro protocols can readily generate hESC and iPSC-derived cells with hematopoietic progenitor function, none of these populations has exhibited what should be the hallmark of an HSC: robust, long-term, multilineage reconstitution of an immunodeficient recipient upon transplantation. These studies address this issue by using transgenic hESC and iPSC lines which report the expression of genes known to be crucial for early hematopoietic events in mice so that they may help us to understand how they translate to human development in vitro. Furthermore, this effort is complemented by additional studies using hESC-derived stromal populations to provide assays that help assess putative HSC quality, maintenance and the hematopoietic niche.