The CD34(+) human acute myeloid leukemia-derived cell line MUTZ-3 is dependent on hematopoietic growth factors for its proliferation and is able to differentiate into dendritic cells (DCs) in response to the combination of granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha. This cell line carries human leukocyte antigen (HLA)-A2.1, HLA-A3, and HLA-B44, which cover most of the caucasian population, and it could therefore be used as an off-the-shelf allogeneic DC-based vaccine. Signal transduction and activation of transcription (STAT) 5b is involved in cytokine signal transduction, particularly of cytokines involved in DC precursor growth and differentiation. The constitutively active form of STAT5b induced cytokine-independent growth of MUTZ-3 cells. Furthermore, STAT5b-transduced cells differentiated into mature DCs in 3 to 4 days after stimulation with DC differentiation-inducing cytokines, reducing the culture period to obtain mature DCs with 5 days compared with unmodified MUTZ-3-derived mature DC cultures. Both DC types expressed DC maturation markers and were equally effective in inducing primary T-cell responses. DCs derived from the STAT5b-transduced cells had a more stable mature phenotype after cytokine deprivation, which was reflected in a better performance in functional assays. In conclusion, these results show that STAT5b-transduced MUTZ-3 can be propagated in cytokine-free medium and rapidly differentiated into functional mature DCs that sustain a mature phenotype over a period of 3 to 5 days in the absence of differentiation-inducing cytokines. The simplified propagation and rapid differentiation into mature DCs may facilitate clinical application of this cell line as an allogeneic DC-based vaccine.