Invasion and metastasis are controlled by the invadopodia, which delivers matrix-degrading enzymes to the invasion interface permitting cancer cell penetration and spread into healthy tissue. We have identified a novel pathway that directs Lyn/Src family tyrosine kinase signals to the invadopodia to regulate sarcoma cell invasion via the molecule AFAP1L1, a new member of the AFAP (Actin Filament Associated Protein) family, the expression of which is strongly associated with malignant osteosarcoma, liposarcoma and leiomyosarcoma (p<0.0001). AFAP1L1 can transform cells, promote migration, and its co-expression with active Lyn profoundly influences cell morphology and movement. AFAP1L1 intersects several invadopodia pathway components through its multiple domains and motifs including two pleckstrin homology domains that bind phospho-lipids generated at the plasma membrane by PI-3 kinase, a direct filamentous-actin binding domain, and two phospho-tyrosine motifs (pY136 and pY566) that specifically bind Vav2 and Nck2 SH2 domains, respectively and which are essential for AFAP1L1-mediated cytoskeleton regulation. Through Vav2 AFAP1L1 regulates Rac activity and its down-stream differential control of PAK1/2/3 (p21-activated kinases) leading to inhibition of myosin light chain kinase (MLCK) and reduced myosin light chain-2 (MLC2) phosphorylation, while Nck2 can recruit actin-nucleating complexes. Significantly, in osteosarcoma cell lines phospho-AFAP1L1 binds Vav2 and Nck2, and knockdown of AFAP1L1 inhibits pMLC2 recruitment to filamentous-actin structures, disrupts invadopodia formation, cell attachment and migration/invasion. These data define a novel pathway that directs Lyn/SFK tyrosine kinase signals to sarcoma cell invadopodia through specific pY-motif mediated recruitment of Vav2 and Nck2 to AFAP1L1 to control cell migration/invasion.