Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, has emerged as a potential oncogene in melanoma. Here, we confirm that Tyro3 is specifically overexpressed in primary melanoma samples and show that Tyro3 is expressed at varying levels in numerous melanoma cell lines. Short hairpin RNA-mediated knockdown of Tyro3 led to significant cell death via apoptotic mechanisms in nearly all melanoma cell lines tested, regardless of the BRAF or NRAS mutation status or co-expression of Axl and/or Mer. We generated soluble and monomeric versions of the human Tyro3 extracellular domain and human Gas6 for affinity measurements and correlated these values with the level of Gas6 required to induce Tyro3 signaling in cellular assays. Calcium was critical for the correct folding of Gas6 and its binding to Tyro3. In melanoma cell lines, Gas6 induced Tyro3 phosphorylation and downstream Akt phosphorylation without apparent effects on Erk. We generated monoclonal antibodies (mAbs) against Tyro3 to examine their effect on survival signaling in melanoma cell lines. The mAbs generated against Tyro3 included nonligand blockers, partial blockers, and competitive ligand blockers. A number of weak and partial ligand blockers (all recognizing the Tyro3 Ig domains) were the most effective at blocking ligand-mediated downstream signaling of Tyro3. Overall, these data indicate that Tyro3 may confer increased survival signals in melanoma cells and can be stymied using inhibitory mAbs. These mAbs may be useful for further investigations of the role of Tyro3 in melanoma.