(A) MPB CD34⁺ HSPC were injected i.v., i.b.m. using a standard syringe, or slow i.b.m. using a Hamilton syringe over 1 minute, and homing of CD34⁺ cells to the BM was analyzed 20–22 hours later. To determine if mechanical pressure in the injected femur alters homing, irradiated CD34^– cells were injected i.b.m., and CD34⁺ cells were injected i.v. into the same animal. Homing was determined in the injected femurs (IF) and non-IF with i.b.m. injections, and in both femurs (BF) combined with i.v. injection. Symbols represent individual animals; n = 3 mice per group; statistical analysis was performed using 2-way ANOVA. (B and C) For i.b.m. injections, BM from the IF and non-IF was analyzed for expression of CXCL12 and VCAM1 on mouse stromal cells (hCD45^–PKH-26^–, mouse lineage^–mCD45^–mCD51⁺ cells). n = 5 mice per group; data were normalized to i.v. injected group; statistical analysis was performed by Mann-Whitney U test. (D and E) CXCR4 and VLA4 homing receptor expression of CD34⁺ HSPC, CD34⁺CD38⁺ HPC, and CD34⁺CD38^–CD90⁺ HSC-enriched populations. Data were normalized to CXCR4 and VLA4 expression on CD34⁺ HSPC. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. (F) Adhesion of CD34⁺ HSPC, CD34⁺CD38⁺ HPC, and CD34⁺CD38^–CD90⁺ HSC to CXCL12. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. (G) Binding of CD34⁺ HSPC, CD34⁺CD38⁺ HPC, and CD34⁺CD38^–CD90⁺ HSC to the VLA4 affinity detection ligand LDV-FITC. Data were normalized to LDV-FITC expression in CD34⁺ HSPC. Symbols represent unique MPB donors; n = 3 per group; statistical analysis was performed using 1-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. In B–E and G, data were normalized to the indicated group, to account for variability in MFI between different experiments/donors.