The acute promyelocytic leukemia (APL) has been treated with all-trans retinoic acid (RA) for decades. While RA has largely been ineffective in non-APL AML subtypes, co-treatments combining RA and other agents are currently in clinical trials. Using the RA-responsive non-APL AML cell line HL-60, we tested the efficacy of the Src family kinase (SFK) inhibitor bosutinib on RA-induced differentiation. HL-60 has been recently shown to bear fidelity to a subtype of AML that respond to RA. We found that co-treatment with RA and bosutinib enhanced differentiation evidenced by increased CD11b expression, G/G cell cycle arrest, and respiratory burst. Expression of the SFK members Fgr and Lyn was enhanced, while SFK activation was inhibited. Phosphorylation of several sites of c-Raf was increased and expression of AhR and p85 PI3K was enhanced. Expression of c-Cbl and mTOR was decreased. Our study suggests that SFK inhibition enhances RA-induced differentiation and may have therapeutic value in non-APL AML.