The BM microenvironment is required for the maintenance, proliferation, and mobilization of hematopoietic stem and progenitor cells (HSPCs), both during steady-state conditions and hematopoietic recovery after myeloablation. The ECM meshwork has long been recognized as a major anatomical component of the BM microenvironment; however, the molecular signatures and functions of the ECM to support HSPCs are poorly understood. Of the many ECM proteins, the expression of tenascin-C (TN-C) was found to be dramatically up-regulated during hematopoietic recovery after myeloablation. The TN-C gene was predominantly expressed in stromal cells and endothelial cells, known as BM niche cells, supporting the function of HSPCs. Mice lacking TN-C (TN-C(-/-)) mice showed normal steady-state hematopoiesis; however, they failed to reconstitute hematopoiesis after BM ablation and showed high lethality. The capacity to support transplanted wild-type hematopoietic cells to regenerate hematopoiesis was reduced in TN-C(-/-) recipient mice. In vitro culture on a TN-C substratum promoted the proliferation of HSPCs in an integrin α9-dependent manner and up-regulated the expression of the cyclins (cyclinD1 and cyclinE1) and down-regulated the expression of the cyclin-dependent kinase inhibitors (p57(Kip2), p21(Cip1), p16(Ink4a)). These results identify TN-C as a critical component of the BM microenvironment that is required for hematopoietic regeneration.