In vitrodifferentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in uteroinitiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls and examples of surface markers used for analysis and cell sorting.