Lymphoid organ stromal cells comprise different subsets whose origin remains unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originated from embryonic mesenchymal progenitors of the Nkx2-5+Islet1+ lineage. This lineage included embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable of supporting ectopic lymphoid-like structures, and a subset of resident spleen stromal cells that proliferated and regenerated the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair, and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity.