Early committed erythroid progenitors (BFU-E to basophilic erythroblasts) separate into 4 transcriptional groups correlating with Ter119 staining intensity. (A) Flow cytometry assessment of marrow cells from wild-type, Flvcr1-deleted, and EPO-treated mice. B220^–Gr1^–CD11b^– marrow cells were divided into populations I-V as before.^15,33  Single erythroid precursors from populations I and II were analyzed as described in the Materials and methods. In wild-type mice, the hemoglobin (HGB) equals 13.8 ± 0.7, and mean corpuscular volume (MCV) equals 44.8 ± 3.1; in Flvcr1-deleted mice, the HGB is 6.2 ± 1.3 and MCV is 72.2 ± 1.9, and in EPO-treated mice, the HGB is 15.9 ± 1.1 and MCV is 53.9 ± 0.9. The percentages of erythroid cells with given Ter119 intensities varied substantially; for example, Ter119^neg cells comprised 0.93%, 4.39%, and 1.69% of cells from wild-type, Flvcr1-deleted, and EPO-treated mice, respectively. (B) Method validation. Transcription (RPKM) of α-globin (Hba-a1) closely correlated with β-globin (Hbb-bs) (r = 0.98), reflecting their coordinate regulation.³⁴  The RNA-to-protein correlation of Glya to Ter119 (r = 0.56) was high, whereas Tfrc to CD71 (r = 0.41) was lower, as anticipated, as transferrin is a highly recycled protein.³⁵  CD44 staining did not correlate with Cd44 expression (r = 0.15), indicating that Cd44 transcription decreases before or very early after erythroid commitment and protein levels and then decreases with successive cell divisions.^33,36  (C) A plot of the Ter119 staining intensity of cells from wild-type mice identified 4 distinct cell groups, labeled A-D. This established the Ter119 staining intensity cutoffs that were then applied to cells isolated from Flvcr1-deleted and EPO-treated mice to identify developmentally equivalent cell groups in an unbiased manner. (D) Normalized Ter119 expression levels of individual cells from wild-type control, Flvcr1-deleted, and EPO-treated mice grouped A-D, using the cutoffs of panel C. Flvcr1-deleted cluster A cells are further separated into A (alive) and Ad (dying) subsets. Mean values ± SD are presented.