Loss of both Gfi1 and Gfi1b reduces the number of HSCs. (A) Flow cytometric analysis of bone marrow cells of pIpC-treated wt, MxCre tg Gfi1b^fl/fl, MxCre tg Gfi1^fl/fl, and MxCre tg Gfi1^fl/fl Gfi1b^fl/fl mice after electronic gating for LSK cells and for the indicated markers. Results for MxCre tg Gfi1^fl/fl Gfi1b^fl/fl were obtained 15 days after the first pIpC injection (4 days after the last pIpC injection). (B) As in panel A, with frequencies depicted in percent with regard to total bone marrow (*P ≤ .05; ***P ≤ .001; n = 14 for wt, n = 14 for MxCre tg Gfi1b^fl/fl and n = 3 for MxCre tg Gfi1^fl/fl). (C) Simultaneous deletion of Gfi1 and Gfi1b reduced the frequency of HSCs in bone marrow by 10-fold approximately 15 days after the first pIpC injection of HSCs (**P ≤ .01). Frequencies of HSCs again reach normal (wt) levels in pIpC-injected MxCre tg Gfi1^fl/fl Gfi1b^fl/fl mice, when measured 40 days after the first pIpC injection (n = 14 for wt, n = 14 for MxCre tg Gfi1b^fl/fl, n = 3 for MxCre tg Gfi1^fl/fl, and n = 3 for MxCre tg Gfi1^fl/fl Gfi1b^fl/fl). (D) Genotyping of sorted HSCs of pIpCinjected MxCre tg Gfi1^fl/fl Gfi1b^fl/fl mice 15 days after the first pIpC injection. Excision of the Gfi1 allele is complete, showing the presence of a functional Cre recombinase, but excision of the Gfi1b allele is incomplete.