Cytokines play a key role in the differentiation, growth and survival of hematopoietic cells in the bone marrow (BM) stroma microenvironment. The mechanisms by which stromal derangements may contribute to the evolution of hematopoietic neoplasias are largely unknown. Here, we characterized BM stromal cells isolated from children with acute lymphoblastic leukemia and determined the effect of the interaction between stromal cells and lymphoblasts on cytokine expression as well as the effect of prednisolone using mono- and co-culture models. The analyses demonstrate that (1) stromal cells and lymphoblasts display different patterns of cytokine gene expression individually. (2) Stromal cells influence gene expression of cytokines in lymphoblasts and vice versa. (3) Glucocorticoid substitution inhibit cytokine gene expression in stromal cells. These findings indicate that stromal cells are important components involved in malignant hematopoiesis and also in response to therapy.