We have examined the effect of oleic acid (OA) concentrations and incubation time, along with chicken serum (CS), on adipogenic differentiation and expression of adipogenic transcripts in hen preadipocytes. Preadipocytes were treated with (i) an adipogenic cocktail (DMI) containing 500 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 20 µg/mL insulin alone and DMI + 75, 150, 300 or 600 µM OA for 48 h; (ii) DMI + 300 µM OA (DMIOA) for 6, 12, 24 or 48 h; and (iii) foetal bovine serum (FBS), CS, DMI + FBS, DMI + CS, DMIOA + FBS and DMIOA + CS. While FABP4 was significantly expressed with increasing concentrations of OA, the expression of C/EBPβ, LEPR and FAS were unchanged compared with the control. PPARγ2 expression was unchanged across all time-points. A significantly higher level of C/EBPα was measured at 48 h, but the levels of C/EBPβ increased after 12 h. Levels of FABP4 significantly increased with the time of incubation after 12 h, but that of LPL was reduced (P < 0.05) at 6, 24 and 48 h. FABP4 was highly expressed in cells treated with CS, DMI + CS and DMIOA + CS compared to cells treated with FBS, DMI + FBS and DMIOA + FBS. In conclusion, increased concentrations of OA and incubation time increases lipid accumulation; FABP4 and C/EBPβ are potential transcription factors regulating OA induced adipogenesis of fat cells obtained from laying hen. CS is a potent inducer of adipogenic differentiation in hen preadipocytes.