Birinapant (1) is a second-generation bivalent antagonist of IAP proteins which is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted auto-ubiquitylation of cIAP1 in vitro. Birinapant-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-B activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as Compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2 and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with: i. decreased potency against cIAP2 and affinity for XIAP BIR3; and, ii. decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity and this improved tolerability has allowed 1 to proceed into clinical studies.