L3MBTL1 knockdown specifically promotes erythroid, but not myeloid or megakaryocytic, differentiation of human CD34⁺ cells. (A) L3MBTL1 expression levels were assessed by quantitative RT-PCR in normal CD34⁺ CB cells. The cells were placed in different culture conditions stimulating erythroid, myeloid, and megakaryocytic differentiation for 2, 5, and 7 days. Total RNA was extracted from 2 × 10⁵ cells. (B) Seventy-two hours after lentiviral infection, GFP⁺CD34⁺ cells were cultured in myeloid conditions for 7, 9, and 11 days, and the expression of myeloid-specific markers CD11b (CD14 and CD33 not shown) was assessed by flow cytometric analysis (n = 3). (C) Seventy-two hours after lentiviral vector infection, the GFP⁺CD34⁺ cells were cultured in megakaryocytic conditions for 7, 9, and 11 days, and expression of the megakaryocytic-specific marker CD41 was assessed by flow cytometric analysis (n = 3). (D) GFP⁺CD34⁺ cells, 72 hours after lentiviral vector infection, were cultured in erythroid conditions for 7, 9, and 11 days, and expression of the erythroid-specific marker GlyA was assessed by flow cytometric analysis (n = 3).