The developmental biology of mammalian B lymphocytes is characterized gby a series of compartmental transitions that begin at the hematopoietic stem cell (HSC) stage, and culminate in immature B cells expressing functional B cell receptors (BCR). Functional heavy (H) and light (L) chain gene rearrangements are the sine qua non for successful transition from one compartment to the next. Targeted disruption of genes that encode components of the pre-BCR or BCR (i.e., H chain genes, surrogate (y) light chain genes), or components of the recombinase ensemble (i.e., RAG-1, RAG-2), all result in mice with severly impaired B cell development. Studies conducted during the last grant period have utilized in vitro models with physiologic context to the human bone marrow (BM) microenvironment to examine the role of cytokines and BM stromal cells in regulating human B cell development. The current proposal builds on this model to address several unresolved issues in mammalian B lymphopoiesis. The specific aims are: 1) to characterize the development of B- lineage cells from common lymphoid progenitors, 2) to identify the BM stromal cell-derived molecules that are essential for regulating the survival and growth of human B cell precursors, and the role of BM stomal cells in cooperating with pre-BCR signaling, and 3) to elucidate the role of Notch family members and their ligands in regulating developmental transitions in human B lymphopoiesis. Multiparameter cell sorting of rare lymphoid progenitors from primary human lymphoid tissue will be evaluated for their growth factor requirements and developmental potential, using freshly isolated human BM stromal cells as a microenvironment. Novel cell lines and antibodies will be used to provide fresh insights into how the pre-BCR functions in B cell development, and the potential role of the Notch family of cell fate detemination protein in regulating key checkpoints in B cell development. The collective results from these studies will enhance our understanding of the normal developmental program of human B lymphocytes and perturbations in the program that can give rise to leukemia and immunodeficiency.