Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase in the insulin receptor super-family. We recently demonstrated that the growth factors pleiotrophin (PTN) and midkine (MK) are ligands for ALK and that upon ALK activation, insulin receptor substrate-1 (IRS-1) and other substrates are phosphorylated. Here, the role of IRS-1 in ligand-mediated ALK signaling is investigated in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. These cells do not express ALK and IRS family members, and do not respond to exogenously added PTN or MK. We show that expression of ALK plus IRS-1 renders these cells independent of IL-3 owing to the activation of ALK by endogenous MK. Mutational analysis reveals that this transformed phenotype of 32D cells requires kinase-active ALK as well as the interaction of ALK with IRS-1. Furthermore, 32D/IRS-1/ALK cells display an enhanced activation of mitogen-activated protein kinase and PI3-kinase pathways, and a selective transcriptional activation of nuclear factor (NF)-κB. Small interfering RNA-mediated knockdown of the endogenous MK or p65/NF-κB revealed that both these are rate limiting for the transformed phenotype induced by ALK plus IRS-1. We conclude that the recruitment of IRS-1 to activated ALK and the activation of NF-κB are essential for the autocrine growth and survival signaling of MK.