The death of Tak1^−/− HSPCs is nearly completely prevented by the inhibition of both RIP-1 and caspase-8 activity. (A-C) c-kit⁺ HSPCs were isolated from Tak1^fx/fx, Tak1^fx/fxTnfr1^−/−r2^−/−, and Tak1^fx/fxFas^−/− mice and separately infected with MSCV-Cre-GFP and MSCV-GFP virus. (A-B) Infected cells were incubated in HSPC medium with or without addition of 30 μg/mL Nec-1. The dynamic changes in GFP⁺ cell ratios were examined at the indicated time points. Data were normalized to MSCV-GFP infected HSPCs. Dashed line represents the normalized data for MSCV-GFP infected HSPCs. **Significant difference between Nec-1–treated groups and vehicle-only groups. (C) Infected HSPCs were purified by FACS 16 hours after infection and incubated in HSPC medium with or without addition of Nec-1. Death of infected cells was analyzed 24 hours later by annexin V staining. (D) WT and Tak1^−/−Tnfr1^−/−r2^−/− mice were injected with poly I:C on days 1 and 2 to induce Tak1 deletion. BM MNCs were isolated on day 3 to examine the activity of caspase-8 and caspase-3 by Western blotting. The bands indicated by arrows are active forms of caspase-8 or caspase-3. (E-G) c-kit⁺ HSPCs were isolated from Tak1^fx/fx and Tak1^fx/fxTnfr1^−/−r2^−/− mice and coinfected with MSCV-Cre-YFP and MSCV-Δ-caspase-8-GFP or MSCV-Δ-FADD-GFP virus. Coinfected cells were purified by FACS to sort for GFP⁺YFP⁺ cells (D) and incubated in HSPC medium for 24 hours with or without Nec-1 treatment. Death of coinfected cells was analyzed by annexin V staining (F-G). MSCV-GFP and MSCV-Cre-YFP infections were studied in parallel as controls. (C,F-G) Data are a representative of 3 independent experiments.