Long-term cultures (LTC) producing dendritic cells (DC) have been previously established from spleen. LTC support the development of nonadherent cells comprising small DC progenitors and immature DC. Similarly, the splenic stroma STX3, derived from a LTC which ceased DC production, can support DC development from precursors in overlaid bone marrow. The STX3 stroma is an immortalised mixed population of endothelial cells and elongated spindle-shaped cells, thought to be fibroblasts. The stromal cell components of STX3 have been studied here. A panel of 102 cell lines was established by single-cell sorting. A range of clone morphology, including cobblestone cells and elongated spindle-shaped cells, was reflective of heterogeneity in STX3. However, similar expression levels for the endothelial genes ACVRL1/ ALK1, COL18A1, and MCAM in 13 splenic stromal cell lines suggested that both cell types had endothelial origin. The hematopoietic support function of stromal clones was tested in coculture assays with a bone marrow cell overlay. Splenic stromal cell lines with different morphology were both supporters and nonsupporters of hematopoiesis, in terms of foci formation or release of suspension cells. Cloning of STX3 led to the isolation of a panel of splenic endothelial cell lines heterogeneous in terms of morphology and hematopoietic support function.