Known HSPC surface markers do not predict ERK signaling dynamics, but signaling dynamics predict future HSPC fates. (A-D) Expression levels of CD33 in HSCs and CD71 in MPPs were quantified for 10 time points before cytokine stimulation (−40 to −4 minutes). Average values for each cell were z-normalized within individual experiments. Pooled data from 3 independent experiments are shown, with symbols representing single cells. CD33 expression levels of HSCs exhibiting different ERK signaling dynamics are shown upon the stimulation of 3F (n = 90 cells) (A) and 5F (n = 72) (B). CD71 expression levels of MPPs are shown in response to 3F (n = 86) (C) and 5F (n = 108) (D). No statistical significance (P > .05) was detected between 4 clusters by pairwise Wilcoxon rank sum test with FDR correction. (E) ERK signaling dynamics in single HSCs upon 5F stimulation. Cells are organized according to the identified 3 clusters (transient, intermediate, and sustained) based on hierarchical clustering (dynamic time warping and Ward’s linkage method) of responding cells (n = 109). Gray bar at the top indicates the timing of cytokine presence. Black solid arrow indicates the addition of cytokines. (F) Population average of ERK signaling dynamics in the 3 clusters identified in E. (G-I) ERK response dynamics predict CD45RA and CD34 expression in HSC daughter cells. Expression of CD45RA, CD33, and CD34 (average over a cell’s lifetime) in HSCs and their progeny was quantified for 3 days after 5F stimulation. Statistical analysis by unpaired Student t test with Welch’s correction. ****P < .0001; ***P < .0005; **P < .005; *P < .05; ns, P > .05.