Anaplastic large cell lymphomas (ALCL) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is fused to several partners, most frequently to the NPM gene. We have previously demonstrated that the constitutive expression and phosphorylation of ALK chimeric proteins is sufficient for cellular transformation, and its activity is strictly required for the survival of ALCL cells. To unravel signaling pathways required for NPM-ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of ALK positive ALCL cell lines through experimentally controlled approaches in which ALK signaling was abrogated by an inducible ALKshRNA or by ALK inhibitors. Transcripts derived from the gene expression profiling analyses uncovered a reproducible signature, which includes a novel group of ALK-regulated genes. A functional RNAi screening identified new ALK transcriptional targets instrumental to cell transformation and/or to sustain the growth and survival of ALK positive ALCL cells. Thus, we prove that an experimentally controlled and functionally validated gene expression profiling analysis represents a powerful tool to identify novel pathogenetic networks and to validate biologically suitable target genes for therapeutic interventions.