Microtubule-targeting agents (MTAs), such as the taxanes and vinca alkaloids, are anti-cancer agents that perturb microtubule function. In cell culture, these drugs exert their cytotoxicity primarily by causing mitotic arrest and cell death. However, accumulated indirect evidence suggests that MTAs may exert their cytotoxicity in human tumors by interfering with interphase microtubules. We therefore sought to develop and characterize a cell system with which to test the hypothesis that MTAs induced interphase death. In primary adult acute lymphoblastic leukemia (ALL) cell cultures treated with vincristine, the major characteristics of mitotic death, which include an increase in cells with 4N DNA content prior to the appearance of cells with <2N DNA content, as well as the co-existence of mitotic and apoptotic markers, were only weakly represented, suggesting a mixed response. The ALL cells were therefore separated into distinct phases of the cell cycle by centrifugal elutriation and labeled with 5-ethynyl-2’-deoxyuridine (EdU) prior to treatment with vincristine. Cells isolated in G1 phase and treated with vincristine underwent cell death without evidence of EdU staining, indicating that they had died without entering S phase and had thus died directly from G1 phase. In contrast, cells isolated in S or G2/M phases underwent death following mitotic arrest. Thus, vincristine induces distinct death pathways in primary ALL cells depending on cell cycle phase context, with cells in G1 phase susceptible to perturbation of interphase microtubules. Studies were undertaken to investigate the possible mechanisms of interphase cell death, including determination of caspase-3 activation and Bax activation, and examination of Bcl-2 protein family expression and MAPK signaling pathways. The results indicate that primary ALL cells represent a powerful model system to study clinically relevant mechanisms of MTA-induced cell death.