Bone marrow mesenchymal stromal cells (MSCs) can rescue acute lymphoblastic leukemia (ALL) cells from l-asparaginase by replenishing the depleted asparagine. As both vincristine (VCR) and imatinib mesylate (IM) can inhibit MSCs' proliferation, we hypothesized that these drugs might reduce the niche support of MSCs to ALL cells. As a consequence, they can help to re-establish the cytotoxic potential of l-asparaginase on ALL cells even under MSCs support. In our study, pre-treating human MSCs with VCR but not IM, markedly reduced the protective capacity of MSCs. Furthermore, differential rescue effects were observed during addition of exogenous l-asparagine to co-culture with or without VCR pre-treatment. This supported the postulation that VCR could suppress the protective effect of MSCs to ALL cells by suppressing l-asparagine secretion. Our results suggested that the combined VCR and l-asparaginase treatment in ALL were synergistic and VCR can serve as an effective agent in suppressing the leukemic marrow microenvironment.