Ex vivo culture system allows temporally controlled deletion of Mcl1 from erythroid cultures. Fetal liver cells were isolated from E12.5 Mcl1^F/F Rosa-ERCreT2⁺ or Mcl1^F/wt Rosa-ERCreT2⁺ embryos; TER119⁻ progenitor cells were purified using MACS MicroBeads and then cultured on fibronectin-coated plates. To induce Mcl1 deletion, fetal liver cells were treated with 4-OHT during the first 18 hours/overnight of culture (t = 0) or overnight the second day of culture (t = 24). Controls did not receive 4-OHT (No 4-OHT). (A) Diagram showing conditions and the time at which 4-OHT was added. (B) Cells were cultured as indicated and then harvested and analyzed by PCR. “Control” indicates PCR control and represents the expected banding pattern; No 4-OHT represents experimental control and represents nondeleted cells. (C) Immunoblot of MCL-1 levels in in vitro–differentiated cells treated with 4-OHT, as indicated. Ponceau S staining was used to indicate loading. (D) Representative flow cytometric analysis of CD45⁻ in vitro–differentiated Mcl1^F/F Rosa-ERCreT2⁺ fetal liver cells. (E) Cytospin preparations from Mcl1^F/wt Rosa-ERCreT2⁺ or Mcl1^F/F Rosa-ERCreT2⁺ fetal liver cells treated with 4-OHT overnight the second day of culture (original magnification ×40, Wright-Giemsa stain). (F) Cells were differentiated as indicated, harvested, and labeled with CD45, CD71, TER119, and Annexin V antibodies. Graph shows the percentage of Annexin V⁻ (live) cells (n > 6 embryos from 2 biological replicates). Data are mean ± standard error of the mean. ****P < .001, 1-way analysis of variance, with α = 0.05. ns, not significant; NT, no treatment.