Bcl-x^−/− definitive erythroid cells hemoglobinize poorly and undergo excessive apoptosis. (A) bcl-x^+/− and bcl-x^−/− definitive erythroid (EryD) precursors were enumerated after replating 10-day-old embryoid bodies into cultures containing Epo and Kit ligand (KL, stem cell factor). Similar results were obtained using two independently derived bcl-x^−/− ES cell clones (nos. 48 and 99). Analyses in B through D were performed using bcl-x^−/−EryD colonies from clone no. 48. Error bars represent standard error of the mean from three separate cultures. (B) bcl-x^−/−EryD colonies are normal in size, but exhibit defective hemoglobinization. Colonies shown are 6 days old. Original magnification × 200. (C) May-Grünwald-Giemsa staining of erythroid cells from 6-day-old bcl-x^+/− and bcl-x^−/− EryD colonies. Note the larger blast-like cells and cells with pyknotic nuclei within bcl-x^−/−colonies. Original magnification × 600. (D) Excessive apoptosis within cells derived from bcl-x^−/− EryD colonies. Upper triangles show DAPI staining (blue), which displays the nuclei of all cells. The same cells were stained for apoptosis using the TUNEL stain (lower triangles, green).