Mechanisms of hematopoietic failure in patients with aplastic anemia (AA) are obscure. We investigate alterations in the hematopoietic microenvironment in AA patients. We present the results of studying mesenchymal stromal cells (MSC), fibroblastic colony-forming units (CFU-F), and adherent cell layers (ACL) of long-term bone marrow cultures (LTBMC) from bone marrow (BM) samples of AA patients. MSC of AA patients proliferated longer than those of donors. In half of the patients' MSC cultures, adipogenesis was impaired. Osteogenic differentiation was not achieved in 36% of AA MSC. CFU-F formed enlarged colonies, and their concentration in the BM of AA patients was significantly increased. Our data suggest that the physiological activation of the stromal microenvironment is characteristic of AA. We detected a decrease in the expression of the angiopoetin-1 (ANG-1) and vascular cell adhesion molecule-1 (VCAM-1) genes, together with an increase in the expression of vascular endothelial growth factor (VEGF) in ACL of AA patients. This indicates abnormal regulatory patterns in both osteoblastic and vascular contexts. Addition of AA patients' serum to donors' LTBMC for 3 weeks induced similar gene expression alterations. The addition of parathyroid hormone (PTH) resulted in the expression levels of analyzed genes returning to normal, in both AA LTBMC and donor cultures treated with AA serum. The physiologic status of the BM stromal microenvironment (MSC, CFU-F, and ACL of LTBMC) of AA patients was altered.