Notch inhibition affects survival and B-cell differentiation of HSPCs. Inhibition of Notch was achieved by addition of 10μM DAPT to CD146⁺ perivascular cells and CB CD34⁺ cell coculture every other day. Vehicle (DMSO) was added to control cocultures. (A-B) Total number of CD45⁺ cells and CD34⁺Lin⁻ cells was significantly reduced after 2 weeks of coculture with DAPT (5.03 ± 0.54 × 10⁴ vs 3.02 ± 0.37 × 10⁴ CD45⁺ cells, n = 4 independent experiments, each experiment was performed in triplicate, **P < .01; 1.5 ± 0.16 × 10⁴ vs 0.82 ± 0.12 × 10⁴ CD34⁺Lin⁻ cells, n = 4 independent experiments, each experiment was performed in triplicate, **P < .01). (C) Similarly, the total number of CFUs was significantly reduced after 4 weeks of coculture with DAPT (478.3 ± 112.4 vs 191.0 ± 43.28, n = 3 independent experiments, each experiment was performed in triplicate; * P < .05). (D) Flow cytometry viability analysis revealed a significantly higher frequency of PI⁺ dead cells in coculture performed in the presence of DAPT (13.08% ± 1.13% vs 19.94 ± 1.31, n = 4 independent experiments, each experiment was performed in triplicate, *** P < .0001). (E) Notch inhibition also significantly increased B-cell development (0.13 ± 0.04 × 10³ vs 1.72 ± 0.55 × 10³ of lymphoid cells, n = 3 individual experiments, each experiment performed in triplicate; **P < .01). Data are presented as mean ± SEM.