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Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) remains challenging due to induction of rapid cellular differentiation after detachment from the supporting bone marrow stem cell niche. However, a few leukemia-related chimeric transcription factors, including MLL fusion proteins, confer remarkable CD34+ ex vivo expansion capacity. In this study, we fused the coding sequence of a FKBP12-derived destabilization domain (DD) to an MLL-fusion gene (MLL-X) and then retrovirally expressed the protein switch in human CD34+ progenitors derived from healthy donors. In the presence of Shield1, a chemical inhibitor of DD fusion protein turnover, the cytokines FLT3-L and IL-3 were necessary and sufficient to yield massive and long-term expansion of DD-MLL-X engineered HSPC-derived late monocytic precursors, with normal karyotype preserved. Upon removal of Shield1, the cells completely lost self-renewal capacity, lost colony-forming properties and spontaneously differentiated, even after 150 days of ex vivo expansion. In the absence of Shield1, a five-day stimulation with IFNγ, LPS, and GM-CSF triggered robust monocytic differentiation. Immunophenotypic characterization revealed upregulation of the monocytic cell surface markers CD14, CD68, CD80, CCR2, and MHC class I and II, in well-accordance with cellular morphology as judged by cytospin preparations. Upregulation of inflammatory markers such as IL-6, IL-10, and CCL2 on mRNA level was detected by qRT-PCR. In functional assays, we finally demonstrated the propensity of the obtained cells to migrate towards the chemokine CCL2, attach to tissue-culture treated plastic surfaces and VCAM-1 under flow and shear stress, to produce reactive oxygen species and engulf both bacterial and cellular particles. Moreover, we demonstrated Fc receptor recognition and phagocytosis of Raji lymphoblastic tumor cells in a daratumumab antibody-dependent manner, proving their identity as monocytes. Taken together, we demonstrate the conversion of a harmful transcription factor to a useful tool for ex vivo blood cell production. Using this engineered protein switch, we were able to produce HSPC-derived late monocytic progenitors in large-scale and differentiate those towards functional monocytes under well-defined ex vivo conditions, both efficiently and quantitatively. This controllable system could set the stage for directed generation of patient-derived monocytes for cell-based immunotherapeutic approaches. [ABSTRACT FROM AUTHOR]