Supplemental Data Figure S1. Cell sorting strategy to purify murine HSC and MPP. Representative gating of HSC and MPP. HSCs are defined as Lin (CD2, CD3, CD4, CD8, Mac1, Gr1, Ter119, B220)-, CD48-, Sca1+, cKit+, CD150+. MPPs are defined as Lin (CD2, CD3, CD4, CD8,Mac1,Gr1, Ter119, B220)-, CD48-, Sca1+, cKit+, CD150-. 10,000 HSC and MPP were simultaneously sorted into 350 μl RLT buffer (QIAGEN) directly for RNA isolation. Figure S2. Ectopic expression of Hoxa5 in mouse fetal liver HSCs. (A). Schematic diagram of transplantation strategy. (B). Transduction efficiency of retro-viral infection of Hoxa5 and GFP control. Fetal liver HSCs (E14.5) were enriched by AutoMACS. After two rounds of spin-infection, transduction rate were detected by flow cytomerty. (C) Real time PCR confirmed the expression pattern of Hoxa5 in GFP positive bone marrow cells of recipient mice. One million GFP positive bone marrow cells of Hoxa5-GFP or GFP control were sorted. 2 μg RNA was reversely transcripted into cDNA using oligo (T) universal primer. 25ng cDNA were used as input of q-RT-PCR(Bio-Rad). Relative fold was calculated by value of 2^(-ΔΔCt). The value of GFP-Ctr was normalized to 1 for comparison purpose. Error bar indicates standard Error. (**P<0.01). (D) Lineages analysis of Peripheral blood of Secondary recipients of Hoxa5-HSCs. Figure S3. Decreased pool of HSC/MPP, and reduced CFU counts of bone marrow cells from recipient mice 6-month posttransplantation. (A) Flow cytometry analysis of HSC and MPP in bone marrow cells isolated from recipients transplanted with Hoxa5-HSC and GFP control. Plots from one representative experiment were shown. (B) Statistic anlaysis of absolute MPPs and HSCs from 100 million bone marrow nucleated cells. **, p<0.01. (C) Colony forming assay indicates that bone marrow cells overexpressing Hoxa5 produce much less colonies in vitro. 40,000 total bone marrow cells or sorted GFP+ bone marrow cells from Hoxa5-GFP and control mice were planted in semisolid medium. The data showed colony numbers counted at Day 8. Error bar means standard error. *, P<0.05; **, P<0.01 (n=3). Gene Symbol Slamf1 Gdf3 Fhl1 Vwf Tjp1 Cdkn1c Apoe Slc6a15 Socs2 Sdc2 Tgm2 Trpc6 Ndn Jam3 Ppic Grb10 Mllt3 Rab27b Tek Dach1 Cttn Transcription per Million HSC MPP 63.94 0.00 22.77 0.00 71.41 7.63 62.89 7.72 9.76 1.32 75.58 11.94 867.43 209.32 26.84 7.55 38.90 11.61 23.77 7.54 307.34 104.91 67.34 23.69 146.00 53.90 44.23 16.50 82.56 35.41 69.49 30.16 176.82 87.31 20.22 10.12 44.36 23.41 46.67 24.76 83.55 47.98 Table S1. RNA-seq results of well-known genes differentially expressed in HSC vs MPP.