The genetic control of cell fate specification, morphogenesis and expansion of the spleen, a crucial lymphoid organ, is poorly understood. Recent studies of mutant mice implicate various transcription factors in spleen development,but the hierarchical relationships between these factors have not been explored. In this report, we establish a genetic network that regulates spleen ontogeny, by analyzing asplenic mice mutant for the transcription factors Pbx1, Hox11 (Tlx1), Nkx3.2 (Bapx1) and Pod1 (capsulin, Tcf21). We show that Hox11 and Nkx2.5, among the earliest known markers for splenic progenitor cells, are absent in the splenic anlage of Pbx1homozygous mutant (^-/-) embryos, implicating the TALE homeoprotein Pbx1 in splenic cell specification. Pbx1 and Hox11genetically interact in spleen formation and loss of either is associated with a similar reduction of progenitor cell proliferation and failed expansion of the splenic anlage. Chromatin immunoprecipitation assays show that Pbx1 binds to the Hox11 promoter in spleen mesenchymal cells, which co-express Pbx1 and Hox11. Furthermore, Hox11 binds its own promoter in vivo and acts synergistically with TALE proteins to activate transcription,supporting its role in an auto-regulatory circuit. These studies establish a Pbx1-Hox11-dependent genetic and transcriptional pathway in spleen ontogeny. Additionally, we demonstrate that while Nkx3.2 and Pod1 control spleen development via separate pathways, Pbx1genetically regulates key players in both pathways, and thus emerges as a central hierarchical co-regulator in spleen genesis.