The Supplementary file contains three figures with figure legends.

1. The Supplementary file contains three figures with figure legends. The files submitted as a 1997-2003 Powerpoint file.

2. a NS 108 107 # DP cells 106 105 Wt >Wt Wt>Dll3-/- b 108 NS 107 #CD4+ cells 106 105 Wt >Wt Wt>Dll3-/- c 108 NS 107 #CD8+ cells 106 105 Wt >Wt Wt>Dll3-/- Supplemental Figure 1. Wild type T cells develop normally in a Dll3-/- host. Irradiation chimeras were set up using bone marrow cells from wild type CD45.1+ donor animals and these were transferred to irradiated Dll3+/+(CD45.2) or Dll3-/- (CD45.2) recipient mice. Twelve weeks after the transfer the thymus and spleen were removed and cells analysed by flow cytometry. The data shows the number of CD45.1+ (a) DP cells, (b) CD4+ and (c) CD8+ T cells in the thymus. Unpaired t test was used to test for statistically significant differences between groups. NS = not significant. Supplemental Figure 1 Hoyne et al

3. Supplemental Figure 2 Hoyne et al Dll3-/- : CD45.1 > insHel Dll3+/+: CD45.1 > insHel a CD4 CD8 Gated on CD4+ cells CD4 1G12 b 35 65 55 45 Gated on DP cells 45 55 30 70 c CD45.1 CD45.1 cells 100 100 CD45.2 cells 10 10 #CD4 1G12+ (x 106 ) #CD4 1G12+ (x 106 ) 1 1 0.1 0.1 0.01 0.01 1 10 100 1 10 100 #DP cells (x106) #DP cells (x106) Supplemental Figure 2. Negative selection of autoreactive CD4+ T cells is not affected by Dll3 deficiency. a. Representative FACS dot plots showing CD4 v CD8 expression of total thymocytes from a Dll3+/+ TCR:TCR+CD45.1+ > insHel mice or Dll3-/- TCR+:TCR+CD45.1+ > insHel mice (top panel). Lower panel shows CD4 v 1G12 staining on the CD4 gated thymocyte population. b. Representative histograms showing the proportion of CD45.1+ and CD45.1-ve cells in the DP or CD4+ 1G12+ gates of representative chimaeras. c. Total cell numbers of DP and CD4+ 1G12+ cells in the thymus of individual chimaeric mice. Cells from the Dll3+/+:CD45.1 chimeras (filled squares), and Dll3-/-:CD45.1 chimaeras (open triangles) are shown.

4. Dll3+/+ 100 100 80 80 60 60 40 40 % T cells % B cells 20 20 0 0 0 0.1 0.3 1 L-685,458concentration (mM) Dll3-/- 100 100 80 80 60 60 % B cells 40 40 % T cells 20 20 0 0 0 0.1 0.3 1 L-685,458concentration (mM) Supplemental Figure 3. T cell production by Dll3-/- precursors is dependent on Notch cleavage by g-secretase. Lineage negative DN cells isolated from the thymus of either Dll3+/+ or Dll3-/- mice were cultured on OP9-Dll1 cells in the presence or absence of the g-secretase inhibitor L-685,458 (Calbiochem, gsi-X) at the indicated concentrations. FACS analysis of Thy1 versus B220 expression was made 8 days after commencement of the culture. The graphs show the frequency of B cells (B220+) (black squares) and T cells (Thy1+) (white squares) arising in the same cultures. Top graph shows the T vs B cell production in Dll3+/+ derived cells and lower graph shows T vs B cells derived from Dll3-/- progenitors. Each point represents the average of 6 replicate wells and is representative of one of three assays performed. Supplemental Figure 3 Hoyne et al