The onset of P-selectin expression occurs postnatally and is regulated by a developmental transition in HSPCs. (A) P-selectin surface expression in unstimulated and stimulated platelets isolated from various gestational ages postnatally. Values shown are expressed as a percent of CD62P MFI on activated adult platelets. Platelets harvested from murine embryos (E12.5), pups (days 1, 7, 14, 21, and 42 after birth), and non-pregnant adult mice, were activated with thrombin (0.5 units/mL) in the presence of antibodies to detect activated αIIbβ₃ (JON/A) and surface P-selectin (CD62P). Significance was determined using a 2-way ANOVA followed by a Bonferroni posttest *P < .01, ** P < .001 versus activated adult P-selectin surface expression; n ≥ 3. (B) Representative histogram of MFI of CD62P (P-selectin) on activated platelets from P1, P14, P42, and adult. Postnatally, a single platelet population transitions from expression low levels of P-selectin (P1) to higher levels of P-selectin (Adult). (C) Experimental scheme. Lin⁻sca⁺kit⁺ (HSPCs) cells were sorted from E14.5 fetal livers and adult bone marrow of UBC-GFP mice and transplanted into sublethally irradiated GFP⁻ recipients. Whole blood from recipients was activated on days 10, 14, 28, and 42 posttransplantation with 0.5 U/mL thrombin for 10 minutes in the presence of antibodies for activated αIIbβ₃ (JON/A) and surface P-selectin (CD62P). HSPCs were taken from E14.5 fetal livers; therefore, days 10 (D10), D14, D28, and D42 posttransplant have equivalent gestational ages of P3.5, P7.5, P21.5, and P35.5, respectively. Donor (GFP⁺) and recipient (GFP⁻) platelet populations were evaluated individually for activated integrin αIIbβ₃ and P-selectin surface expression. (D) Integrin activation (αIIbβ₃) in untreated and thrombin-treated donor (GFP⁺) platelets. Comparable amounts of integrin activation were seen in platelets produced from both bone marrow (BM)–derived and fetal liver (FL)–derived donor HSPCs on days 10, 14, 28, and 42 after transplantation. Analysis performed using 2-way ANOVA with Bonferroni posttest, n ≥ 3. (E) P-selectin surface expression in untreated and thrombin-treated donor (GFP⁺) platelets. On days 10 and 14 posttransplant, FL-derived HSPCs produce platelets with low levels of P-selectin surface expression after activation relative to activated donor platelets from BM-derived HSPCs. By D28 posttransplant, platelets produced from either BM-derived HSPCs or FL-derived LSKs have comparable P-selectin expression after activation. Platelets produced by BM-derived HSPCs maintain comparable levels of P-selectin surface expression on all days after transplantation, suggesting developmental changes in FL HSPC’s result in the production of platelets with gradually increasing levels of P-selectin surface expression. Significance was determined using a 2-way ANOVA followed by a Bonferroni posttest *P < .001; n ≥ 3. (F) Integrin activation (αIIbβ₃) in untreated and thrombin-treated recipient (GFP⁻) platelets. Comparable amounts of integrin activation were found on platelets of the recipient mouse after transplantation of bone marrow or fetal-liver derived LSKs. (G) P-selectin surface expression in untreated and thrombin-treated recipient (GFP⁻) platelets. Comparable amounts of P-selectin surface expression were found on platelets of the recipient mouse after transplantation of BM- or FL-derived LSK (Lin⁻sca⁺kit⁺). No significance was detected using a 2-way ANOVA with Bonferroni posttest, n ≥ 3.