Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia.