Spleen stromal cells are critical determinants of dendritic cell (DC) development in spleen. The spleen stromal line, namely STX3, supports DC differentiation in vitro from overlaid bone marrow cells while the lymph node stromal line, namely 2RL22, does not. Here we have characterised the hematopoietic support capacity of each stroma, and analysed lineage origin of the stromal cell lines by gene profiling using microarrays. Stromal co-culture experiments were performed using bone marrow cells as a source of hematopoietic progenitors. A characteristic immature myeloid-like CD11c(+)CD11b(+)CD86(+)MHC-II(-/lo)B220(-)CD8 alpha-DC is produced after 14 days in STX3 cocultures, while 2RL22 cocultures produce only monocyte/macrophage-like cells. No other hematopoietic cell type is produced. The STX3 and 2RL22 stroma were compared by transcriptome analysis utilising Affymetrix Murine U74Av2 genechips to identify gene expression related to differential hematopoietic support function. Data mining was used to determine cell surface marker expression reflecting endothelial cells and fibroblasts, as well as adhesion molecules contributing to the microenvironment. STX3 shows gene expression reflective of early endothelial cells, while 2RL22 expresses markers specific to fibroblasts. The expression of genes like Flt1, CD34, Mcam, and Eng distinguishes STX3 as an early immature endothelial cell lacking markers of angioblasts or hemangioblasts like Tal1/SCL, Tie1, Tie2, Kdr or Prom1/AC133. The absence of expression of genes like Vwf and Cd31 distinguishes STX3 from fully differentiated vascular endothelial cells. In contrast, the 2RL22 lymph node stroma specifically expresses genes related to fibroblastic-like cells like osteoblasts with expression of Vdr (Vitamin D receptor), and epithelial cells with expression of Krt13 (keratins). Gene expression data identifies STX3 as splenic endothelial cells, independently able to support the outgrowth of immature, myeloid DC-like cells from progenitors present in bone marrow, while 2RL22 lymph node fibroblastic cells provide support for development of monocytes/macrophages.