Umbilical cord blood (UCB) T cells can be redirected to kill leukemia and lymphoma cells by engineering with a single-chain chimeric antigen receptor (CAR) and thus may have general applications in adoptive cell therapy. However, the role of costimulatory molecules in UCB T-cell activation and effector functions in context with CAR remains elusive. To investigate the effect of costimulatory molecules (4-1BB and CD28) on UCB T cells, we transduced UCB T cells with lentiviral vectors expressing Green Fluorescent Protein (GFP) and CAR for CD19 containing an intracellular domain of the CD3zeta chain and either a 4-1BB (UCB-19BBzeta) or a CD28 intracellular domain (UCB-1928zeta), both (UCB-1928BBzeta), or neither (UCB-19zeta). We found that UCB-19BBzeta and UCB-28BBzeta T cells exhibited more cytotoxicity to CD19(+) leukemia and lymphoma cell lines than UCB-19zeta and UCB-1928zeta, although differences in secretion of interleukin-2 and interferon-gamma by these T cells were not evident. In vivo adoptive transfer of these T cells into intraperitoneal tumor-bearing mice demonstrated that UCB-19BBzeta and UCB-1928BBzeta T cells mounted the most potent antitumor response. The mice adoptively transferred with UCB-1928BBzeta cells survived longer than the mice with UCB-19BBzeta. Moreover, UCB-1928BBzeta T cells mounted a more robust antitumor response than UCB-19BBzeta in a systemic tumor model. Our data suggest a synergistic role of 4-1BB and CD28 costimulation in engineering antileukemia UCB effector cells and implicate a design for redirected UCB T-cell therapy for refractory leukemia.