Hematopoiesis occurs throughout the lifespan of an organism. This process involves the formation of blood cells from self-renewing hematopoietic stem cells (HSC). The hematopoietic niche environment, which comprises non-hematopoietic stromal cells, regulates the quiescence, dormancy, self-renewal and differentiation of HSC. It is known that multiple HSC niches including endosteal, vascular and perivascular exist in the bone marrow, although niches in other sites like spleen are only starting to be defined. Previous studies in this lab have described unique splenic stromal cell lines 5G3 and 3B5 which can support restricted hematopoiesis in vitro reflecting HSC niches. Inhibition assays were also used to identify the important role of SCF and Notch but not C-XC motif ligand 12 (CXCL12) in supporting in vitro hematopoiesis. Transcriptome data previously showed that 5G3 and 3B5 express many genes in parallel with perivascular cells described in bone marrow. The study here shows that both 5G3 and 3B5 stroma share lineage origin with perivascular cells in bone marrow. 5G3 and 3B5 express cell surface markers in common with these cells including CD105, CD29, VCAM1, Sca-1, CD51, CD140a and Thy1.2. In addition, freshly isolated splenic stromal cells with the Sca-1+gp38+Thy1.2+CD29+CD51+ phenotype, reflecting cells of mesenchymal lineage were found to be important for in vitro hematopoiesis. Stromal cells expressing gp38 or Thy1.2 are also associated with HSC in spleen identified through section staining. Restricted hematopoiesis giving rise to specific types of myeloid cells was replicated in vivo following grafting of splenic stromal cell lines 5G3, 10C9 and 3B5 under the kidney capsule. This study has improved our understanding of HSC niches in spleen and identified several molecular regulators of hematopoiesis. Information obtained will be important for development therapies involving splenic or ectopic niches to enhance hematopoietic output, in immunocompromised patients or following HSC transplantation.